CUNY Brooklyn College Effects of THP 1 Derived Exosomes On HepG2 Cells

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8263045 7029BMS Laboratory Book Overall Aim: The overall aim of this investigation was to determine whether exosome-mediated communication between macrophages and HepG2 liver cancer cells can induce changes in cell phenotype and behaviour. This was done utilising flowcytometry, qPCR and migration assay techniques. The hypothesis was that inducing exosome-mediated communication between macrophages and HepG2 cells would increase cell migration and proliferation. Moreover, sample 2 would exhibit the largest change in cell phenotype and behaviour as it contained the middle fraction of exosomes which was the purest, least contaminated and most concentrated fraction. Session 1: Exosome Purification The initial phase of this investigation was the purification of exosomes from THP-1 monocytic, cell line, throughout which the aim was to isolate a sample of exosomes with maximum yield and maximum purity (Patel et al., 2019). Furthermore, the aim was to obtain the 3 fractions containing the highest concentration of exosomes which we estimated to be fractions 9-11. This was accomplished utilising Sepharose CL-2B column to perform size-exclusion chromatography (SEC) which involves the use of a porous stationary phase and a mobile phase (Lobb & Möller, 2017). The nature of the stationary phase allows for the differential elution of particles from the solution; bigger particles elute first, followed by smaller vesicles (Sidhom et al., 2020). Differential ultracentrifugation (dUC) has traditionally been the gold standard for exosome purification. dUC relies on the sequential separation of particles by density- and sizedependent sedimentation using a series of centrifugation steps (Konoshenko et al., 2018). Most dUC procedures require up to 3 hours of centrifugation as an increase in centrifugation time is directly correlated with exosome purity. As such, in recent years other techniques such as SEC have become preferable as they are less labour intensive and produce purer samples(Konoshenko et al., 2018). Although SEC is overall the preferred method of exosome purification, if this investigation were to be repeated it would be suggested that a Sepharose CL-4B column be used as a 2020 study by Benedikter et al. 2017, found that an increase in the percentage of agarose present in the 4B column provided better separation of small particles such as exosomes when the mobile phase was cell culture media (Benedikter et al., 2017). PKH67 Staining After the initial purification of exosomes, the three fractions were stained using PKH67, a lipophilic fluorescent label. The aim of this technique was to successfully label the exosomes to enable their presence to be detected when reading at the appropriate wavelength. 8263045 PKH67 is a green fluorochrome with excitation at 490nm which functions by intercalating aliphatic reporter molecules into the lipid bilayer. This mechanism of intercalation is unspecific therefore, it is imperative that the sample of exosomes be pure to ensure accurate results (Pužar Dominkuš et al., 2018). Although this technique is currently the most common method of staining exosomes, there is new evidence that suggests due to its unspecific nature there are instances where it also stains non-plasma membrane cellular components such as lipo-proteins (Simonsen, 2019). If I were to repeat this investigation, I would continue to use the same PKH67 staining technique. This is because the alternative methods of staining are either not cost effective, such as transfection of cells to produce exosomes containing a genetically encoded fluorescent reporter such as CD63-eGFP (Sutaria et al., 2017), or it impairs the functionality of exosome through the staining of a less dynamic transmembrane-bound exosome proteins (van der Vlist et al., 2012). RNA Extraction The aim of this technique in this investigation was to extract high quality, high yield exosomal RNA from the isolated fractions in preparation for use in RT-qPCR. In this investigation the Trisure method of RNA extraction was used, as such thiocynate and phenol compounds were used to facilitate the disruption of cells during homogenisation. Subsequently, chloroform was used to separate the homogenate into 3 phases where RNA is found in the upper aqueous phase. The RNA was then washed in multiple steps to remove impurities. Although this is a relatively cost-effective method with a high yield, it requires significant manual processing and as such is time-intensive when compared to simpler techniques such as the column separation method (Scholes & Lewis, 2020). Subsequently, if I were to repeat this investigation, I would use the spin column method which is an RNA extraction method that utilises membranes than contain silica to bind to nucleic acids. Lysates pass through the silica membrane using centrifugal force with the RNA binding to the silica until it is eluted with RNase-free water. This method is simple and involves very little manual labour, moreover due to depleted chances of ethanol contamination samples are of higher quality (Mônica Ghislaine Oliveira et al., 2016). Wound Healing Assay Wound healing assay is an assay performed in 2D cell monolayer where a cell-free region is created through the deliberate destruction of the confluent cell monolayer, such as by a pipette tip as in this investigation, which is then available for cells to occupy (Stamm et al., 2016). The gathering of data across numerous time points is able to demonstrate the progression of cell proliferation (Cory, 2011). In this investigation the aim of this method was to determine the effects of exosome-mediated communication on the behaviour of HepG2 liver cancer cells through a change in cell growth patterns. The wound-healing assay, as performed in this investigation has numerous advantages over more complicated methods as it is easy to carry-out and all materials are widely available in any lab. However, utilising a pipette tip to create the cell-free gap causes irregular scratches and therefore difficulty ensuring reproducibility and comparability of results (Liang et al., 8263045 2007). As such, if I were to complete this investigation again, I would utilise silicon insert to ensure uniformity across all cell-free gaps. In addition to this, I would introduce more timepoints at which cell-migration would be measured in order to elucidate greater data trends. Reflection and Planning for Next Session: Overall, I feel as if the first session was relatively positive. I was nervous to be back in the lab after so long, but this led to me being very prepared having read and annotated the protocols in great detail. Despite working well in a group, with so many people in the lab I felt rushed to get in and out of the cell culture hood which meant I did not use good aseptic technique as I would usually. Hopefully this did not impact my results and has not led to any contamination of my samples. Next session my goal is to stay calm and take my time with the methods whilst still working efficiently. Session 2: Reverse Transcription Reverse transcription involves the production of cDNA from an RNA template via the enzyme reverse transcriptase. Small RNA cDNAs are generated by reverse transcription (RT) utilising microRNA (miRNA)-specific RT primers which detect only the mature miRNA target and not precursor molecules (TaqManTM MicroRNA Reverse Transcription Kit, 2018). In this investigation the aim was to utilise the TaqMan MicroRNA Reverse Transcription Kit to produce a high yield, high quality cDNA for use in real time quantitative polymerase chain reaction (RT-qPCR). The TaqMan MicroRNA Reverse Transcription Kit is highly specific and well suited to miRNAs due to the inclusion of the RT stem-loop primer and as such is able to accurately convert miRNA to cDNA while maintaining quality and yield. If I were to repeat this method, I would use the same TaqMan miRNA kit due to its specialisation for microRNAs (TaqManTM MicroRNA Reverse Transcription Kit, 2018). Flowcytometry The aim of the flowcytometry technique used in this investigation was to detect the percentage uptake of exosomes and subsequent change in cell phenotype of HepG2 liver cancer cells. Flowcytometry involves the passage of cells in single file through the path of a laser. Cell components are fluorescently labelled and excited by the laser to emit light which can then be detected. The exosomes purified in this investigation were stained with PKH67 fluorescent dye, and as such, cells that displayed exosome uptake exhibited fluorescence at the FITC-A wavelength (Rim & Kim, 2016). If I were to repeat this investigation, I would continue to utilise flow cytometry as although exosomes themselves lie outside of the detection limit for flowcytometry, the purpose of this study was to observe exosome uptake and change in cell phenotype. I would suggest the additional use of NanoSight analysis of exosomes as this would elucidate the mean concentration and mean size of exosomes and thus, further data trends could be interpreted. 8263045 Real Time Quantitative Polymerase Chain Reaction (RT-qPCR) RT-qPCR was carried out in this investigation with the aim of quantifying the amount of miRNA, miR-150 within each sample which is commonly found within exosomes derived from THP-1 cells. Moreover, this technique was used to analyse trends in exosome uptake and subsequent exosome-mediated communication between macrophages and HepG2 liver cancer cells and the resultant changes in cell phenotype and behaviour. Within RT-qPCR, the TaqMan probe method and SYBR Green fluorescent dye method are the two most common techniques for miRNA detection. SYBR Green is relatively unspecific and subsequently is unable to recognise non-specific products such as primer dimers which reduces the accuracy of qPCR quantification. if I were to repeat this investigation I would continue to use TaqMan probe RT-qPCR (Ye et al., 2019). Results: Flow Cytometry When PKH67 stained exosomes were incubated with HepG2 cells, a change in the FITC-A fluorescence can be seen. Cells within the M2 gated parameters, as represented in red in figure 1, align with the phenotype of the negative control (figure 1D) which contained cells that had both no PKH67 stain and no exosome, as signified by its peak positioned further left on the X-axis, closer to 103. In contrast, cells within the M3 gated parameters, similarly represented on in figure 1, align with the phenotype of the positive control (figure 1E) which contained cells that exhibited FITC-A fluorescence, and thus were positive for both the PKH67 Stain and exosomes. This is signified by a shift in the peak to the right of X-axis closer to 105. Figure 1B established that sample 2 contained the highest percentage of exosome uptake with 35.7% falling within the M3 parameters, thus exhibiting FITC-A fluorescence representative of PKH67 staining. Figure 1A shows that sample 1 had the next highest percentage with 32.5% falling within the M3 parameters, thus demonstrating FITC-A fluorescence representative of PKH67 staining and exosomal uptake. Therefore, Sample 3, as shown in figure 1C displayed the smallest percentage within the M3 parameters with only 25.7% exhibiting exosomal uptake. 8263045 Figure 1: Detection of FITC-A fluorescence in HepG2 cells incubated with PKH67 stained THP1 derived exosomes by flow cytometry. A) example data for a representative flow cytometry plot of cells treated with fraction 9 exosomes, B) example data for a representative flow cytometry plot of cells treated with fraction 10 exosomes, C) example data for a representative flow cytometry plot of cells treated with fraction 11 exosomes, D) Negative control of unstained cells, E) Positive control of stained exosomes Wound Healing The results presented in table 1 demonstrate an exosome-dependent increase in the percentage reduction in migration area, when compared to the average of T0 values, as seen by positive percentage difference values in column 4. Sample 2 displayed the greatest reduction in migration area when compared to the control at T48 with a value of 66.5%. Sample 1 showed the next greatest percentage reduction in migration area at T48 when compared to the control at 30.6%, followed by sample 3 with 29.5%. Sample 1 2 3 Control (%) 26.8 10.6 17 Exosome Treated (%) 57.3 77.1 46.4 Difference between Control and Exosome Treated (%) 30.6 66.5 29.5 Table 1: Percentage reductions (1 d.p) in cell-free area calculated from mean of T0 values for three samples of example data. 8263045 qPCR As can be seen from figure 2, the CT value for all three samples differed which suggests a difference in their miR-150 content. However, due to variances in conditions such as volume of intact RNA and reaction efficiency, the CT values themselves are unable to accurately represent the true concentration of miR-150 within each sample. However, as shown by the data in figure 3, the CT values were processed utilising U6 snRNA CT values and the 2ΔΔCT equation in order to produce the relative fold changes in each sample. As such, the levels of miR-150 expression were able to be more accurately analysed and compared. The ΔΔCT values shown in figure 3 established that sample 2 had a 1.15-fold increase in miR150 expression when compared to sample 1 indicating that sample 2 had the most miR-150 expression. Conversely, sample 3 was determined to have a 2.14-fold decrease in miR-150 expression when compared to sample 1 therefore indicating that sample 3 had the least miR150 expression across all sample. Figure 2: qPCR Curves for HepG2 cells incubated with 3 separate fractions of purified THP-1 exosomes. All samples were analysed in duplicate for the presence of miR-150 and the housekeeping gene U6. 8263045 1.6 1.4 ΔΔ CT Value 1.2 1 0.8 0.6 0.4 0.2 0 Sample 1 Sample 2 Sample 3 Sample Number Figure 3: ΔΔCT values with standard deviation error bars for HepG2 cells incubated with 3 separate fractions of purified THP-1 exosomes. Discussion and Data Analysis: Exosomes are small extracellular vesicles composed of lipid bilayer with the primary function of mediating cell-cell communication. Due to their highly heterogenous nature, they have recently received significant attention for their role in pathobiological processes and as such, were the focus of this investigation (Weidle et al., 2017). Throughout this study, flowcytometry, RT-qPCr and migration assay techniques were used to determine whether exosome-mediated communication between macrophages and Hep2 liver cancer cells could induce changes in cell phenotype and behaviour. Overall, the results of the techniques used in this investigation revealed an exosomedependent change in both cell phenotype and behaviour. The results of the flowcytometry revealed that all samples exhibited uptake of exosomes which represents a change in cell phenotype. As shown in figure 1, sample 2 exhibited the most substantial change in phenotype, followed by samples 1 and 3 respectively. Although the uptake of exosomes was relatively low, it was significant enough for the results of the migration assay to reveal a direct correlation between the percentage of HepG2 cells that exhibited exosomal uptake in each sample in the flowcytometry, and the percentage reduction in cell-free area in the migration assay, with sample 2 having the largest reduction followed by samples 1 and 3 respectively. Therefore, it can be concluded that exosomalcommunication between macrophage-derived exosomes and HepG2 liver cancer cells causes an increase in cell migration and proliferation and thus a change in cell behaviour. This is supported by a 2021 study by Wu et al., which established that macrophage derived exosomes facilitated tumorigenesis and metastasis by transferring αMβ2 integrin to tumor cells (Wu et al, 2021). The results of the RT-qPCR carried out in this investigation similarly supported the correlation between the percentage of HepG2 cells that exhibited exosomal uptake in each sample in the flowcytometry and the fold changes seen in miR-150 expression. miRNAs are small non- 8263045 coding RNAs which function as regulators of gene expressed that are usually encapsulated in exosomes to ensure the molecules stability (MacFarlane & R. Murphy, 2010). miR-150 normally functions in haematopoiesis where it regulates genes that have key roles in in differentiating stem cells towards becoming megakaryocytes. However, in malignancies, this control over cell differentiation implicates miR-150 as a key promotor in tumour development and cancer progression. Therefore, the increase in migration and cell proliferation seen in the wound-healing assay in addition to the changes in cell phenotype can be attributed to the transport of miR-150 into cells via exosomes. (Zhang et al., 2010) Taken as a whole, the results of this investigation emphasise the range of biological effects associated with exosomes which is a current focus within scientific literature. However, there is significant research required to elucidate both the specific pathways, and the roles that these molecules play in cancer cells. The outcomes of this research have the potential to aid in the development new treatments for previously fatal malignancies (Pritchard et al., 2020). Reflection: I am satisfied with my performance within the lab as I was able to gain experience in both Flowcytometry and RT-qPCR which are both techniques that I haven’t previously carried out. In addition to this, I feel as if I worked more calmly and efficiently in the second lab which was aided by my understanding of the protocol. I was disappointed that I was unable to obtain any viable wound-healing assay results as the cells in my exosome-treated well had become contaminated due to my poor aseptic technique and rushing from the previous lab. In the future, when completing labs such as these again, I will endeavour to stay calm and not rush to maintain proper aseptic technique and avoid sample contamination. Overall Discussion: Overall, the results of this investigation support the proposed hypotheses of an exosomedependent change in cell phenotype/behaviour and that the sample treated with the second fraction of exosomes would result in the greatest change in cell phenotype/behaviour. Moreover, the study fulfilled its aims of demonstrating that exosome-mediated communication between macrophages and HepG2 liver cancer cells would initiate changes in cell phenotype such as; uptake of exosomes and an increase miR-150 expression and changes in cell behaviour such as; increase in proliferation and migration. The conclusion that exosome-mediated communication influences liver cancer progression as shown in studies such as Wu et al., who demonstrated that cell growth and proliferation and subsequently tumour metastasis increased in liver cancer when exposed to macrophagederived exosomes both in vivo and in vitro. However, due to the heterozygous nature of exosomes, the exact pathways through which exosomes cause these outcomes is still unknown (Wu et al., 2021). Exosomes also have the potential to occupy key roles in new treatments as an in vitro study by Liang et al. 2018, found that engineered exosome delivery of active miR-26a to HepG2 liver cancer cells resulted in decreased rates of cell migration and proliferation (Liang et al., 2018). Word Count: 2193 8263045 References: Benedikter, B. J., Bouwman, F. G., Vajen, T., Heinzmann, A. C. A., Grauls, G., Mariman, E. C., Wouters, E. F. M., Savelkoul, P. H., Lopez-Iglesias, C., Koenen, R. R., Rohde, G. G. U., & Stassen, F. R. M. (2017). Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies. Scientific Reports, 7(1). https://doi.org/10.1038/s41598-017-15717-7 Chen, Y., Song, Y., Du, W., Gong, L., Chang, H., & Zou, Z. (2019). 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Molecular Cell, 39(1), 133–144. https://doi.org/10.1016/j.molcel.2010.06.010 Journal of Extracellular Vesicles ISSN: (Print) 2001-3078 (Online) Journal homepage: https://www.tandfonline.com/loi/zjev20 Single-step isolation of extracellular vesicles by size-exclusion chromatography Anita N. Böing, Edwin van der Pol, Anita E. Grootemaat, Frank A. W. Coumans, Auguste Sturk & Rienk Nieuwland To cite this article: Anita N. Böing, Edwin van der Pol, Anita E. Grootemaat, Frank A. W. Coumans, Auguste Sturk & Rienk Nieuwland (2014) Single-step isolation of extracellular vesicles by size-exclusion chromatography, Journal of Extracellular Vesicles, 3:1, 23430, DOI: 10.3402/ jev.v3.23430 To link to this article: https://doi.org/10.3402/jev.v3.23430 © 2014 Anita N. Böing et al. View supplementary material Published online: 08 Sep 2014. Submit your article to this journal Article views: 16067 View related articles View Crossmark data Citing articles: 249 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=zjev20 æ ORIGINAL RESEARCH ARTICLE Single-step isolation of extracellular vesicles by size-exclusion chromatography Anita N. Böing1*, Edwin van der Pol1,2, Anita E. Grootemaat1, Frank A. W. Coumans1,2, Auguste Sturk1 and Rienk Nieuwland1 1 Department of Clinical Chemistry, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands; 2Department of Biomedical Engineering and Physics, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands Background: Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively. Aim: To develop a single-step protocol to isolate vesicles from human body fluids. Methods: Platelet-free supernatant, derived from platelet concentrates, was loaded on a sepharose CL-2B column to perform size-exclusion chromatography (SEC; n3). Fractions were collected and analysed by nanoparticle tracking analysis, resistive pulse sensing, flow cytometry and transmission electron microscopy. The concentrations of high-density lipoprotein cholesterol (HDL) and protein were measured in each fraction. Results: Fractions 912 contained the highest concentrations of particles larger than 70 nm and plateletderived vesicles (46%96 and 61%92 of totals present in all collected fractions, respectively), but less than 5% of HDL and less than 1% of protein (4.8%91 and 0.65%90.3, respectively). HDL was present mainly in fractions 1820 (32%92 of total), and protein in fractions 1921 (36%92 of total). Compared to the starting material, recovery of platelet-derived vesicles was 43%923 in fractions 912, with an 8-fold and 70-fold enrichment compared to HDL and protein. Conclusions: SEC efficiently isolates extracellular vesicles with a diameter larger than 70 nm from platelet-free supernatant of platelet concentrates. Application SEC will improve studies on the dimensional, structural and functional properties of extracellular vesicles. Keywords: extracellular vesicles; isolation; lipoproteins; plasma; protein; size-exclusion chromatography Responsible Editor: Aled Clayton, Cardiff University, UK. *Correspondence to: Anita N. Böing, Department of Clinical Chemistry, Academic Medical Centre, (room B1-238), Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands, Email: [email protected] To access the supplementary material to this article, please see Supplementary files under Article Tools online. Received: 25 November 2013; Revised: 31 July 2014; Accepted: 5 August 2014; Published: 8 September 2014 he scientific and clinical interest in plasma-derived vesicles is tremendous, since these vesicles may contain clinically relevant information (15). Isolation of vesicles from plasma with good recovery and without contamination of proteins and lipoproteins, however, is a challenge. Thus far, most isolation protocols are based on differential centrifugation. After removal of cells in the first low-speed centrifugation step, vesicles are isolated using centrifugal accelerations of 19,000 100,000 g (6). Unfortunately, protein aggregates are gene- T rated at high velocities of 100,000 g, (79) and vesicles may clump. Isolation of vesicles from plasma is further hampered by the viscosity and density of plasma, and by the presence of lipoprotein particles with a density and diameter similar to the extracellular vesicles of interest (6,10,11). Consequently, isolation of vesicles from plasma or serum by density-gradient ultracentrifugation results in co-isolation of high-density lipoprotein (HDL), and isolation of HDL results in co-isolation of vesicles [as described in the response of Yuana Y. and Nieuwland R. Journal of Extracellular Vesicles 2014. # 2014 Anita N. Böing et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 3.0 Unported License (http://creativecommons.org/licenses/by-nc/3.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Citation: Journal of Extracellular Vesicles 2014, 3: 23430 – http://dx.doi.org/10.3402/jev.v3.23430 1 (page number not for citation purpose) Anita N. Böing et al. to (12) and by Vickers K.C. et al. (13)]. Thus, there is an urgent need for a simple and fast protocol to isolate vesicles from human plasma. In platelet research, two different protocols are commonly applied to isolate platelets from platelet-rich plasma, that is, to replace the plasma by buffer. In one procedure, platelets are isolated from the plasma by centrifugation and washing (14,15). In the other procedure, platelets are isolated by size-exclusion chromatography (SEC), also known as ‘‘gel filtration’’ (15,16). SEC has also been used previously to isolate vesicles from sera, ascites and saliva, and was shown to separate vesicles from proteins (1720). Whether SEC separates vesicles from HDL, however, has never been investigated. In this study, we investigate the efficacy of single-step SEC for isolation of extracellular vesicles from human platelet-free supernatant of platelet concentrates and we studied the separation of vesicles by SEC from HDL and proteins. Material and methods Platelet concentrates Platelet concentrates were from Sanquin (Amsterdam, The Netherlands). Buffy coats from 5 whole blood units are pooled together with one plasma unit of one of these donors. Subsequently, this pool was gently centrifuged. The resulting platelet rich plasma was slowly extracted using an automated separator via leukocyte reduction filter to a PVC-citrate storage bag. Platelet concentrates (n3, 912 days old,B1 leukocyte/3 108 platelets) were stored with agitation at room temperature until use. Platelet-depleted plasma Platelet concentrate (30 mL) was diluted 1:1 with filtered phosphate-buffered saline [PBS; 1.54 mol/L NaCl, 12.4 mmol/L Na2HPO4, 2.05 mmol/L NaH2PO4, pH 7.4; 0.22 mm filter (Merck chemicals BV, Darmstadt, Germany)]. Next, 12 mL acid citrate dextrose (ACD; 0.85 mol/L trisodiumcitrate, 0.11 mol/L D-glucose and 0.071 mol/L citric acid) was added and the suspension was centrifuged for 20 minutes at 800 g, 208C. Thereafter, the vesiclecontaining supernatant was isolated and centrifuged (20 minutes at 1,550 g, 208C) to remove remaining platelets. This centrifugation procedure was repeated for 3 cycles, to ensure complete removal of platelets. SEC column Sepharose CL-2B (30 mL, GE Healthcare; Uppsala, Sweden) was washed with PBS containing 0.32% trisodiumcitrate (pH 7.4, 0.22 mm filtered). Subsequently, the tip of a 10 mL plastic syringe [Becton Dickinson (BD), San Jose, CA] was stuffed with nylon stocking (20 denier, Hema, Amsterdam, The Netherlands), and the syringe was stacked with 10 mL washed sepharose CL-2B (Fig. 2) to create a column with diameter of 1.6 cm and height of 6.2 cm (Fig. 1). 2 (page number not for citation purpose) Fig. 1. Image of size-exclusion chromatography column. A 10 mL syringe stacked with sepharose CL-2B for isolation of vesicles from platelet-free supernatant of platelet concentrates. Collection of fractions Platelet-free supernatant of a platelet concentrate (1.5 mL) was loaded on the column, followed by elution with PBS/ 0.32% citrate (pH 7.4, 0.22 mm filtered). The eluate was collected in 26 sequential fractions of 0.5 mL (Fig. 1). For each fraction, the number of particles was determined by nanoparticle tracking analysis (NTA), resistive pulse sensing (RPS) and flow cytometry. In addition, HDL cholesterol and protein concentrations were measured for each fraction. Of each fraction, 200 mL was frozen in liquid nitrogen and stored at 808C for subsequent transmission electron microscopy (TEM) on thawed fractions. Nanoparticle tracking analysis The concentration and size distribution of particles in collected fractions was measured with NTA (NS500; Nanosight, Amesbury, UK), equipped with an EMCCD camera and a 405 nm diode laser. Silica beads (100 nm diameter; Microspheres-Nanospheres, Cold Spring, NY) were used to configure and calibrate the instrument. Fractions were diluted 101,000-fold in PBS to reduce the number of particles in the field of view below 200/image. Of each fraction, 10 videos, each of 30-seconds duration, were captured with the camera shutter set at 33.31 ms and the camera gain set at 400. All fractions were Citation: Journal of Extracellular Vesicles 2014, 3: 23430 – http://dx.doi.org/10.3402/jev.v3.23430 Single-step isolation of extracellular vesicles analysed using the same threshold, which was calculated by custom-made software (MATLAB v.7.9.0.529). Analysis was performed by the instrument software (NTA 2.3.0.15). Resistive pulse sensing The concentration and size distribution of particles was measured with RPS (qNano; Izon Science Ltd, Christchurch, New Zealand) using an NP200A nanopore. This nanopore was suitable for the detection of 100400 nm particles. Samples were measured with 7 mbar pressure, 45 mm stretch and 0.34 V. Samples were analysed for 5 minutes or until 1,000 vesicles were counted, whichever came first. To calibrate size and concentration, carboxylated polystyrene beads (Izon Science Ltd) were sonicated for 10 seconds, diluted in PBS with 0.3 mM sodium dodecyl sulphate and analysed immediately after dilution. Flow cytometry To detect platelet vesicles, 20 mL of each fraction was incubated for 15 minutes with an antibody against glycoprotein IIIa (CD61), which is a subunit of the platelet fibrinogen receptor and also known as integrin b3, phycoerythrin (PE)-conjugated CD61; 5 mL 1:10 prediluted in PBS/citrate, Pharmingen, San Diego, CA). IgG1-PE (BD) was used as control antibody. To detect all vesicles, 20 mL of each fraction was labelled with lactadherin (fluorescein isothiocyanate-conjugated, 5 mL 1:10 prediluted in PBS/citrate). After incubation, 300 mL PBS/citrate was added and samples were analysed on a FACSCalibur (BD, Cellquest version 4.0.2) for 1 minute at a flow rate of 60 mL min 1. The trigger was set on FSC at E00, SSC voltage of 329, threshold FSC 30, SSC 0. No gates were used to determine extracellular vesicles. Transmission electron microscopy After thawing, samples from all fractions, both undiluted and 50-fold diluted in PBS, were subjected to overnight fixation, in 0.1% final concentration (v/v) paraformaldehyde (Electron Microscopy Science, Hatfield, PA). Then, a 200-mesh formvar and carbon coated copper grid (Electron Microscopy Science) was placed on a 10 mL droplet to allow adherence of particles to the grid (7 minutes, room temperature). Thereafter, the grid was transferred onto drops of 1.75% uranyl acetate (w/v) for negative staining. Each grid was studied using a transmission electron microscope (Fei, Tecnai-12; Eindhoven, the Netherlands) operated at 80 kV using a Veleta 2,000 2,000 sidemounted CCD camera and Imaging Solutions software (Olympus, Shinjuku, Tokyo, Japan). Protein The protein concentration was determined using a Bradford protein assay according to the manufacturer’s instructions (Pierce, Rockford, IL). The absorbance was measured at 595 nm on a Spectramax Plus (Molecular Devices, Sunnyvale, CA). In addition, to directly visualize the relative presence of proteins in the collected fractions, in a single experiment, 10 mL of each fraction was mixed with 10 mL 2-fold concentrated reducing sample buffer, boiled for 5 minutes and loaded on an 816% gradient gel (BioRad, Hercules, CA). Proteins were stained with Bio-Safe Coomassie G-250 Stain (BioRad). Western blot Proteins from all fractions (800 mL) were precipitated using trichloroacetic acid (20% final concentration; Sigma-Aldrich, St. Louis, MO). From each fraction, equal amounts of protein (4 mg) were dissolved in nonreducing sample buffer, boiled and loaded on 816% gradient PAGE gels (Biorad), and proteins were transferred to PVDF membrane (Millipore, Billerica, MA). Blots were incubated with anti-CD63 (BD, clone H5C6) or anti-CD9 (BD, clone M-L13), extensively washed and then incubated with a goat-anti-mouse (GAM)-horseradish peroxidase (Dako, Glostrup, Denmark). Subsequently, the PVDF membranes were incubated with a 5-fold diluted peroxidase substrate (LumiLight, Roche Diagnostics, Almere, The Netherlands) for 5 minutes, followed by analysis of luminescence using a LAS4000 luminescence image analyser (Fuji, Valhalla, NY). High density lipoprotein HDL Cholesterol was determined using the colorimetric reagent HDL-Cholesterol Plus third generation (Roche Diagnostics, Almere, The Netherlands) on a Cobas C8000 analyser (Roche) as per manufacturer’s instructions. This assay specifically detects HDL-associated cholesterol (21). Furthermore, a specific protein present in HDL, apo lipoprotein A1 (APO A1), was measured on an Architect (Abbott, Abbott Park, IL) according to manufacturer’s instructions. Recovery and enrichment Recovery was defined as the total number of CD61exposing vesicles in all fractions combined divided by the total number of CD61-exposing vesicles in the starting material. Recovery in a limited number of fractions is the total number in those fractions divided by the total number in the starting material. The enrichment factor of vesicles to protein or HDL in fraction X is the ratio of CD61-exposing vesicles to protein or HDL in fraction X compared to the ratio of vesicles to protein or HDL in the starting material. Results from 3 independent experiments are presented as the mean9the standard deviation. Results Particles by NTA The concentration of particles was determined by NTA in both starting material and fractions. Particles detected by Citation: Journal of Extracellular Vesicles 2014, 3: 23430 – http://dx.doi.org/10.3402/jev.v3.23430 3 (page number not for citation purpose) Anita N. Böing et al. NTA are not necessarily extracellular vesicles. With our settings, NTA will detect single particles with a diameter larger than 70 nm, which may include not only vesicles, but also protein aggregates, chylomicrons [size range 1002,000 nm (22)] and very low density lipoproteins [VLDL; 27200 nm (22)]. NTA will not detect HDL [7 12 nm (22)], low density lipoproteins [LDL; 1823 nm (22)] and intermediate density lipoproteins [IDL; 2327 nm (22)]. After SEC, the highest concentration of particles was found in fractions 912 (Fig. 2a). The recovery of particles measured by NTA was 76%938, and 46%96 of the recovered particles were present in fractions 912. Particles by RPS The concentration of particles was determined by RPS in both the starting material and in the fractions. Also particles detected by RPS are not necessarily vesicles. With our settings, RPS can detect single particles with a diameter of approximately 100400 nm, which will include vesicles, protein aggregates, chylomicrons and VLDL. RPS will not detect HDL, LDL, or IDL. After SEC, the highest concentrations of particles were present in fractions 912 (Fig. 2b). The recovery of particles measured by RPS was 60%910, and 72%91 of the recovered particles were present in fractions 912 (Fig. 2b). Detection of platelet-derived vesicles Since the particles detected in fractions 912 by both NTA and RPS are not necessarily vesicles, we applied flow cytometry to distinguish vesicles from lipoprotein particles. Because the studied extracellular vesicles originated from platelets, we used CD61 and lactadherin as vesicle markers. With our settings, the flow cytometer detects vesicles with a diameter larger than 500 nm. The recovery of CD61-exposing vesicles by flow cytometry was 71%935, and 61%92 of the recovered vesicles were present in fractions 912 (Fig. 2c, for dot plots see Supplementary Fig. 1). The recovery of CD61-exposing vesicles in fractions 912 was 43%923 of the starting material. The recovery of lactadherin-binding vesicles was 163%955, and 44%95 of the recovered vesicles were present in fractions 912 (Fig. 2d). The recovery of lactadherin-binding vesicles in fractions 912 was 73% 931 of the starting material. Lipoproteins and protein The recovery of HDL cholesterol was 103%911, and fractions 1820 contained 32%92 of total recovered HDL (Fig. 2e). Although the HDL cholesterol assay is specific for HDL, we confirmed these measurement results by also measuring APO A1, a specific HDL protein, in a control experiment. The recovery of HDL APO A1 was 72% and fractions 1820 contained 38% of total recovered HDL APO A1 (Fig. 2f). The recovery of protein was 95%917, and fractions 1921 contain 4 (page number not for citation purpose) 36%92 of total recovered protein (Fig. 2g). Fractions 912 contained the majority of vesicles and additionally contained 4.8%91 of total recovered HDL cholesterol, whereas HDL APO A1 was below the detection limit (0.01 g/L) in these fractions. Fractions 912 contained 0.65%90.3 of total recovered protein. Overview of detected parameters Figure 2h shows an overlay of the percentage of particles (NTA, RPS), vesicles (CD61-exposing, lactadherinbinding), HDL (cholesterol, APO A1) and protein per fraction. Particles and vesicles showed a peak at fraction 10, whereas HDL (cholesterol and APO A1) and protein showed a peak at fraction 19 and 20, respectively. Thus, it is clear that vesicles can be separated from protein and lipoproteins by SEC. Presence of proteins per fraction To directly visualize the efficacy of SEC to separate vesicles from plasma proteins, a control experiment was performed in which similar volumes from all collected fractions (as described in Material and Methods) were compared for the presence of plasma protein after gel electrophoresis. The starting material, platelet-free supernatant of a platelet concentrate (1.5 mL), contained very high concentrations of proteins including albumin (66 kDa) when applied directly to gel electrophoresis (3 and 20 mL, Fig. 3a, right gel). Evidently, after SEC low levels of protein become detectable from fraction 8 or 9 onwards, but the bulk of the protein elutes from fraction 15 onwards (Fig. 3a). Thus, vesicles, which are mainly present in fractions 912, are clearly separated from the bulk of soluble plasma proteins by SEC. Presence of CD63 and CD9 per fraction To confirm the detection of vesicles by flow cytometry, we performed a control experiment to study the presence of CD63 and CD9, both vesicle-associated tetraspanins, by Western blot. CD63 and CD9 were both detectable in fractions 9 and 10 (Fig. 3b and c, respectively), confirming the presence of vesicles in these fractions. Visualization of vesicles, lipoproteins and proteins TEM was used to confirm the presence of vesicles or lipoprotein particles. Figure 4 shows representative images of the starting material and fractions 5, 9, 10, 11, 17, 18, 19 and 20 (Fig. 4ai). In the starting material, vesicles were not visible due to the abundant presence of lipoproteins and proteins (Fig. 4a). To improve the visualization of the contents of the starting material and fractions 1720, we also performed TEM on 50-fold diluted samples (Fig. 4jn). In the diluted starting material, vesicles (cup shaped) as well as lipoproteins (white spheres) and proteins (white ragged structures) were visible (Fig. 4j). In fraction 5, no vesicles or lipoproteins are detectable (Fig. 4b), which confirms the Citation: Journal of Extracellular Vesicles 2014, 3: 23430 – http://dx.doi.org/10.3402/jev.v3.23430 Single-step isolation of extracellular vesicles Fig. 2. Presence of vesicles, protein and lipoproteins per fraction. The concentration of vesicles, protein and lipoproteins was measured in each fraction. Each bar shows the number present in a fraction as % of the total number that passed the column. The height of the bar represents the mean, the error bars the standard deviation from 3 experiments. a) Particles (larger than 70 nm) measured by NTA. b) Particles (100400 nm) measured by RPS. c) CD61 vesicles measured by flow cytometry. d) Lactadherinvesicles measured by flow cytometry. e) HDL (Cholesterol) concentration measured by a colorimetric assay. f) HDL (APO A1) concentration measured by a turbidimetric assay. g) Protein concentration measured by a Bradford protein assay h) Overview of all measured results. Citation: Journal of Extracellular Vesicles 2014, 3: 23430 – http://dx.doi.org/10.3402/jev.v3.23430 5 (page number not for citation purpose) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Start 3 µL Marker kDa a) Fraction Fraction Start 20 µL Anita N. Böing et al. 250 150 100 75 50 37 25 20 15 5 6 7 8 9 10 11 12 13 150 100 75 150 100 75 50 50 37 37 25 25 CD63 Fraction 1 2 3 4 5 6 7 8 9 10 11 12 13 150 150 100 75 100 75 50 50 25 20 Fraction 14 15 16 17 18 19 20 21 22 23 24 25 26 Pos. control 10 Start 10 Marker 15 kDa 15 37 CD9 17 18 19 20 21 22 23 24 25 26 20 20 c) 14 15 16 Pos. control 4 Fraction Start 3 Marker 2 kDa 1 kDa CD63 Fraction Marker kDa b) Marker 10 37 25 20 15 15 Fig. 3. Presence of proteins, CD63 and CD9 in collected fractions. a) The presence of proteins in each fraction determined by loading 20 mL on PAGE gels. The molecular weight of albumin is 66 kDa. b,c) Presence of tetraspanins in the different fractions was studied by Western blot, with 4 mg protein used per fraction. First, the presence of CD63 was shown (53 kDa, panel b), and next the presence of CD9 was shown (24 kDa, panel c). The tetraspanin bands are indicated by arrows in panels b and c. Platelet lysate was used as positive control. 6 (page number not for citation purpose) Citation: Journal of Extracellular Vesicles 2014, 3: 23430 – http://dx.doi.org/10.3402/jev.v3.23430 Single-step isolation of extracellular vesicles a) b) c) starting material fr. 5 fr. 9 d) e) f) fr. 10 fr. 11 fr. 17 g) h) i) fr. 18 fr. 19 fr. 20 j) k) l) starting material (50 x) fr. 17 (50 x) fr. 18 (50 x) m) n) fr. 19 (50 x) fr. 20 (50 x) Fig. 4. TEM images of fractions. Starting material and fractions, undiluted or 50-fold diluted when indicated, were analysed by TEM for the presence of particles. All images shown are representative images for the starting material (a, j) and fractions 5, 9, 10, 11, 17, 18, 19 and 20 (bi, kn). Scale bar is 200 nm (ag, jn), 500 nm (h), or 1 mm (i). Citation: Journal of Extracellular Vesicles 2014, 3: 23430 – http://dx.doi.org/10.3402/jev.v3.23430 7 (page number not for citation purpose) Anita N. Böing et al. a) Vesicles Protein HDL Recovery (%) 75 60 45 30 15 0 9 9–10 9–11 9–12 Fraction b) 1000 Enrichment (vesicle to protein) Recovery and enrichment As mentioned, most particles and vesicles were present in fractions 912. To gain insight into the extent of purification of the vesicles, we calculated the recovery of CD61-exposing vesicles, protein and HDL cholesterol of fractions 912 compared to the starting material (Fig. 5a). In fraction 9, the recovery of vesicles was 14%99 and the recoveries of protein and HDL cholesterol were low, 0.023%90.01 and 0.8%90.07, respectively. Thus, sepharose CL-2B SEC results in a 5629337-fold enrichment of vesicles compared to proteins in the starting material (Fig. 5b), and a 17911-fold enrichment of vesicles compared to HDL cholesterol (Fig. 4c). When fractions 9 and 10 were combined, the recovery of vesicles was 31%919, but this increase was at the expense of an increased contamination with protein and HDL cholesterol compared to fraction 9 alone (Fig. 5a). Nevertheless, combining fractions 9 and 10 gives a 3309111fold enrichment of vesicles compared to protein (Fig. 5b), and a 19911-fold enrichment of vesicles compared to HDL cholesterol (Fig. 5c). Combining fractions 911 recovered 38%921 of the vesicles from the starting material and results in a 152937-fold enrichment of vesicles compared to protein (Fig. 5b), and a 1294-fold enrichment of vesicles compared to HDL cholesterol (Fig. 4c). Combining fractions 912 recovered 43%923 of vesicles from the starting material and give a 70919-fold enrichment of vesicles compared to protein (Fig. 5b), and a 89 3-fold enrichment of vesicles compared to HDL cholesterol (Fig. 5c). Thus, it is clear that the recovery of vesicles can be improved by combining fractions 911 or 912, but this result is at the expense of more contamination by protein and HDL cholesterol (Fig. 5a). SEC has several major advantages compared to differential centrifugation and density-gradient ultracentrifugation, which are the most widely applied protocols for vesicle isolation. Compared to differential centrifugation, there is no risk of protein complex formation and vesicle aggregation. In addition, the high viscosity of plasma affects the recovery of vesicles isolated by differential centrifugation (23,24), but does not affect the recovery of vesicles by SEC. Compared to density-gradient ultracentrifugation, buffers with physiological osmolarity and viscosity can be used. The most commonly applied density gradient for the isolation of vesicles, sucrose (6,7,2529), may have some additional downsides. For example, isolation of 800 600 400 200 0 9 9–10 9–11 Fraction 9 9–10 9–11 Fraction 9–12 c) 40 Enrichment (vesicle to HDL (cholesterol)) results of NTA, RPS and flow cytometry. As expected, in fraction 9 the vesicles were clearly visible but also visible were low numbers of lipoproteins (Fig. 4c). Vesicles were also visible in fractions 10 and 11 (Fig. 4de), but the number of lipoprotein particles was increased compared to fraction 9. The vesicles in fractions 911 range in diameter from 70 to 500 nm. Very few vesicles and an abundance of proteins and lipoprotein particles were visible in fractions 1720 (Fig. 4fi undiluted and 4kn 50-fold diluted). Thus, TEM confirms that SEC separates vesicles from proteins and lipoproteins. 30 20 10 0 Discussion We demonstrate that vesicles can be purified from human platelet-free supernatant of platelet concentrates by sepharose CL-2B SEC. With this approach, vesicles can be easily separated from proteins and HDL. We also isolated vesicles from human plasma with SEC, which resulted in similar recoveries of vesicles, proteins and lipoproteins in fractions 912 (data not shown). 8 (page number not for citation purpose) 9–12 Fig. 5. Recovery and enrichment. The recovery and enrichment relative to the starting material, in the vesicle-containing fractions (9, 910, 911, 912) are shown. a) Recovery of vesicles, protein and HDL (cholesterol) in the vesicle-containing fractions. b) Enrichment factor of vesicle to protein. c) Enrichment factor of vesicle to HDL (cholesterol). Citation: Journal of Extracellular Vesicles 2014, 3: 23430 – http://dx.doi.org/10.3402/jev.v3.23430 Single-step isolation of extracellular vesicles organelles by sucrose density-gradient ultracentrifugation is detrimental, since these gradients are ‘‘highly viscous and grossly hyperosmotic, leading to slow sedimentation rates for small particles and loss of water from subcellular organelles’’ (30). Furthermore, whereas several investigators reported a loss of biological function when vesicles are isolated by sucrose density-gradient ultracentrifugation (ISEV meeting Budapest, October 2013), vesicles from saliva isolated by SEC are still fully functional with regard to their capacity to induce coagulation (data not shown, personal communication C.M. Hau), indicating that the biological properties of vesicles seem unaffected after isolation by SEC. Moreover, by density-gradient ultracentrifugation, contaminants with overlapping densities cannot be isolated. For example, the density of HDL considerably overlaps with vesicles (6,10). The recovery of vesicles isolated with SEC is 43%923, when combining fractions 912. Similar recoveries are reported after isolation of vesicles by (ultra) centrifugation and detection by flow cytometry, namely 5080% (31). Furthermore, Momen-Heravi showed a recovery of 2% of plasma vesicles after ultracentrifugation as measured by NTA (23). In both studies, however, contamination by proteins and lipoproteins was not studied. When combining fractions 912, a reduction of 70-fold in the protein to vesicle ratio and 8-fold in the HDL to vesicle ratio is found. To our knowledge, it is unknown to which extent HDL and protein are reduced in the vesicle fraction after a (sucrose gradient) ultracentrifugation protocol. However, despite the reduced contamination after isolation of vesicles with SEC, we recommend the use of blood samples collected from fasting subjects, to minimize potential contamination by chylomicrons and VLDL. The principle of SEC is separation based on a difference in size. The sepharose beads in the column have pores with a diameter of approximately 75 nm (32,33), and a tortuous path through the bead. A particle that can enter the beads is delayed due to the increased path length. All particles larger than 75 nm, including lipoproteins, cannot enter the beads and can only travel along with the void volume fluid. Based on our TEM results, the smallest vesicles that are present in fraction 912 have a diameter of 70 nm or larger, which confirms the theoretical separation of components below and above 75 nm. Because the size distribution of vesicles, as measured by RPS and NTA, does not change between the starting material and fraction 912 (data not shown), there seems to be no separation of vesicles in the size range from 70 to 500 nm. We confirmed this by making a mixture of 100 and 400 nm silica beads. After application of SEC, 45 48% of both sizes of beads were recovered in fractions 912, confirming no separation within this size range (data not shown). Vesicles smaller than 75 nm are probably present in the fractions high in HDL, that is fractions 1820. From the size distributions of vesicles in urine (34) and erythrocyte concentrates (Y. Yuana, personal communication), we estimate that approximately 50% of all vesicles are larger than 75 nm. It is unknown whether vesicles smaller than 75 nm harbour different clinical information than the larger vesicles. Sepharose CL-2B has relatively large pores. Choice of a sepharose with smaller pores may allow the isolation of vesicles smaller than 75 nm, albeit with higher contamination by lipoproteins. We used sepharose CL-2B in a 10 mL plastic syringe (Fig. 1), which has a diameter of 1.6 cm and a height of 6.2 cm. We expect that the column height, column diameter and sample volume can be optimized to improve separation of vesicles from contaminants and the recovery of vesicles. For example, a longer, narrower column with the same volume of sepharose may result in an improved separation of protein and vesicles. A narrower column with a smaller volume of sepharose and the same length, on the other hand, may result in a higher recovery of vesicles. Investigators should optimize those parameters to their own experimental needs. Because the size distribution of vesicles does not vary between fractions 9 and 12, we assume that the vesicles in each fraction are comparable. Fraction 9 is the purest vesicle fraction, but contains only 14% of the vesicles in the starting material. The method used for further analysis determines whether it is best to collect only fraction 9 or to combine multiple fractions. For example, for proteomics the lowest possible contamination with protein is essential and the use of fraction 9 only may be optimal. For TEM imaging, the background is much improved when comparing fractions 911 to the starting material (Fig. 4ad). Combining multiple fractions may result in a higher density of vesicles on the TEM grid, speeding up the analysis. For flow cytometry, we prefer a higher concentration of vesicles if this does not lead to swarm detection (35,36), and thus fractions 912 would be combined. In conclusion, our study shows that vesicles of a diameter larger than 75 nm can be isolated from complex body fluids such as plasma by single-step SEC. Purification of vesicles in combined fractions 9-12 relative to protein and HDL is 70- and 8-fold, respectively. Recovery of vesicles with sepharose CL-2B SEC is 43% compared to 280% with ultracentrifugation. Thus, compared to ultracentrifugation, SEC results in a good recovery of vesicles with almost complete removal of contaminants. Furthermore, vesicle isolation by sepharose CL-2B SEC takes less than 20 minutes, compared to 296 hours for ultracentrifugation, thus vesicle samples can be prepared for analysis on the same day of collection. In addition, sepharose CL-2B SEC components cost approximately t15,- and no expensive equipment is needed. Thus, isolation of vesicles by SEC is quick, cheap and easy. 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Lipoprotein subclasses and particle sizes and their relationship with coronary artery calcification in men and women with and without type 1 diabetes. Diabetes. 2002;51:194956. 23. Momen-Heravi F, Balaj L, Alian S, Trachtenberg AJ, Hochberg FH, Skog J, et al. Impact of biofluid viscosity on size and sedimentation efficiency of the isolated microvesicles. Front Physiol. 2012;3:1627. 24. O’Brien JR. Cell membrane damage, platelet stickiness and some effects of aspirin. Br J Haematol. 1969;17:6101. 25. Aalberts M, van Dissel-Emiliani FM, van Adrichem NP, van Wijnen M, Wauben MH, Stout TA, et al. Identification of distinct populations of prostasomes that differentially express prostate stem cell antigen, annexin A1, and GLIPR2 in humans. Biol Reprod. 2012;86:8290. 26. Keller S, Ridinger J, Rupp AK, Janssen JW, Altevogt P. Body fluid derived exosomes as a novel template for clinical diagnostics. J Transl Med. 2011;9:8695. 27. Palma J, Yaddanapudi SC, Pigati L, Havens MA, Jeong S, Weiner GA, et al. MicroRNAs are exported from malignant cells in customized particles. Nucleic Acids Res. 2012;40: 912538. 28. Poliakov A, Spilman M, Dokland T, Amling CL, Mobley JA. Structural heterogeneity and protein composition of exosomelike vesicles (prostasomes) in human semen. Prostate. 2009; 69:15967. 29. Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, et al. B lymphocytes secrete antigenpresenting vesicles. J Exp Med. 1996;183:116172. 30. Ford T, Graham J, Rickwood D. Iodixanol: a nonionic isoosmotic centrifugation medium for the formation of selfgenerated gradients. Anal Biochem. 1994;220:3606. Citation: Journal of Extracellular Vesicles 2014, 3: 23430 – http://dx.doi.org/10.3402/jev.v3.23430 Single-step isolation of extracellular vesicles 31. Jayachandran M, Miller VM, Heit JA, Owen WG. Methodology for isolation, identification and characterization of microvesicles in peripheral blood. J Immunol Methods. 2012;375: 20714. 32. Williams A, Hagel L. Column handbook for size exclusion chromatography. Philadelphia, PA: Academic Press; 1999. 33. Hagel L, Östberg M, Andersson T. Apparent pore size distributions of chromatography media. J Chrom A. 1996;743: 3342. 34. van der Pol E, Hoekstra AG, Sturk A, Otto C, van Leeuwen TG, Nieuwland R. Optical and non-optical methods for detection and characterization of microparticles and exosomes. J Thromb Haemost. 2010;8:2596607. 35. Nolan JP, Stoner SA. A trigger channel threshold artifact in nanoparticle analysis. Cytometry A. 2013;83:3015. 36. van der Pol E, van Gemert MJ, Sturk A, Nieuwland R, van Leeuwen TG. Single vs. swarm detection of microparticles and exosomes by flow cytometry. J Thromb Haemost. 2012;10: 91930. Citation: Journal of Extracellular Vesicles 2014, 3: 23430 – http://dx.doi.org/10.3402/jev.v3.23430 11 (page number not for citation purpose) 7029BMS Lab Schedule Analysis of the effects of THP-1 derived exosomes on HepG2 cells Exosomes are microscale extracellular vesicles that are present in most somatic fluids including blood and urine, as well as the media of cell cultures. These-lipid-bearing microvesicles range in size from 30 to 200 nm. They often contain key biomolecules like proteins and nucleic acids (both DNA and RNA) derived from their cell of origin. Recent studies support that exosomes are involved with intercellular communication, including between macrophages and cancer cells. E.g. https://pubs.rsc.org/en/content/articlelanding/2020/ra/c9ra07332a#!divAbstract and https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290460/ and https://www.cell.com/molecular-cell/fulltext/S1097-2765(10)00451X?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1097276510004 51X%3Fshowall%3Dtrue#secd5383008e932 Exosomes therefore likely have important roles in health and various diseases. Below is a set of procedures that have been put together to investigate if exosome-mediated communication between macrophages and liver cancer cells can induce changes in cell phenotypes/behaviour. In general, the instructions will be less detailed than those you followed in 7027BMS, as you are expected to take charge of your own preparation for lab sessions. You will need to read the required information before each session as well as seek additional information (and watch online videos if needed) to ensure you understand what you need to do before coming into the lab. The 7029BMS labs are designed to provide you with an experience similar to that you would encounter in a research lab, where you are expected to be independent and able to plan experiments yourself, often by adapting published procedures to your needs. Session 1 Purification of exosomes (based on the protocol develop by https://www.tandfonline.com/doi/full/10.3402/jev.v3.23430) Work in a group of 3 1. Collect a flask of THP-1 cells – this a non-adherent cell line that produces exosomes. These cells were cultured in a 5 ml culture flask in normal media (RPMI 1640 + 2 mM Glutamine + 10% Foetal Bovine Serum) before being treated with 0.5 nM phorbol myristate acetate (PMA) for 3 h and then placed by back in normal media for a further 24 h. 2. Mix the cells and culture media in the flask gently and transfer to a clean centrifuge tube, spin the tube (with a suitable balance) for 5 minutes at 2000rpm to pellet the cells from the supernatant. 3. Gently pour off the supernatant to a new tube and dispose of the cell pellet in the appropriate discard container. Place your tube with the supernatant on ice. 4. Take the provided 10 ml of sepharose solution (sepharose CL-2B washed with PBS-citrate 0.32% (w/v) pH 7.4, 0.22 µm filtered) and gently swirl to resuspend. Gently pour the solution steadily into the provided syringe (capped at the end using parafilm) and allow to settle. NOTE – step 4 may have been done for you. 5. As described in the publication above, load 1.5 ml of your supernatant onto the column, and elute with PBS-citrate 0.32% (w/v) immediately collecting fractions of a suitable amount (see paper link above to find this information) in eppendorf tubes. Make sure all tubes are labelled with fraction number. Keep the fractions on ice. 6. Add around 5 ml more PBS-citrate 0.32% (w/v) to the column to wash it and let the flow through drip into a waste beaker. You can move to the next step while this drips through. Once the dripping has stopped, cap the end and add more PBS-citrate 0.32% (w/v) to prevent the column from drying out. 7. Pick 3 fractions you think will contain exosomes (according to the paper above). 8. Each member of the group will then pick and work with one of the 3 fractions from now on. Make sure your fraction tube is labelled with your name and fraction number. 9. Transfer 0.25 ml of your fraction to another Eppendorf tube. This will be used for RNA extraction. Make sure this tube is also labelled with your name and fraction number and RNA. 10. Give your 2 tubes to the academic who will store them at -80ºC until required. Session 2 Each student within the group from session 1 should process one of the fraction samples PKH67 staining of THP-1 derived exosomes 1. You will have a tube on your desk containing 1.5 µl of the dye PKH67. Add 0.2 ml of DILUENT C to this tube and mix the tube gently. 2. Add 200 µl of the PKH67/DILUENT C mix to 200 µl of the fraction supernatant you identified that you think will contain exosomes and gently mix continually for 30 seconds using a 1 ml pipette (and blue tip). Incubate your tube at room temperature for 5 minutes. 3. Quench by adding 0.4 ml 1% BSA in PBS. Incubate your tube at room temperature for 5 minutes. Incubation of HepG2 cells with THP-1 exosomes for flow cytometry analysis 1. Using the Cat 2 cabinets, add 0.4 ml of your labelled fraction to a flask of HepG2 cells. Control flasks where nothing is added will be analysed alongside your test samples on (i.e. you do not need to pick up a control flask). Clearly label your flask. 2. Incubated at 37ºC until a later session where they will be analysed by flow cytometry Incubation of HepG2 cells with THP-1 exosomes for scratch wound healing assay 1. Using the Cat 2 cabinets, add the remaining 0.4 ml of your labelled fraction to a well within a 6-well plate of HepG2 cells. A 2nd well will act as a control. Clearly label the wells. The other 4 wells will be used by other students (i.e. there will be 3 students per 6-well plate). 2. Using a sterile 100 µl pipette tip (usually yellow), scratch a straight line down the middle of each well. Make sure to use a different pipette tip for each sample. 3. Have a look at your plate under the microscope and take an image of each scratch (Time 0 timepoint). An academic will be at the microscope to show you how to do this. 4. The plates will then be incubated 37ºC and images will be taken for you over the next 48hr. Session 3 You will use the TaqMan MicroRNA Assay to assess the levels of miR-150 in your chosen exosome sample. Read information from below links: TaqMan Small RNA Assays Quick Reference Card: http://tools.thermofisher.com/content/sfs/manuals/cms_083619.pdf Further information: https://www.thermofisher.com/document-connect/documentconnect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFSAssets%2FLSG%2Fbrochures%2Fcms_078025.pdf&title=QXJ0aWNsZXMgJmFtcDsgV2hpdGUgU GFwZXJzOiBEZXNpZ24gUGlwZWxpbmUgZm9yIFRhcU1hbiZyZWc7IFNtYWxsIFJOQSBBc3Nh eXMgKFRlY2hOb3RlcyBhcnRpY2xlKQ== General guide: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/4364031_TaqSmallRNA_UG.pdf TaqMan™ MicroRNA Reverse Transcription Kit: https://www.thermofisher.com/order/catalog/product/4366596#/4366596 TaqMan® Universal PCR Master Mix II, no UNG: https://www.thermofisher.com/order/catalog/product/4440043#/4440043 TaqMan™ MicroRNA Assay: https://www.thermofisher.com/order/catalog/product/4427975#/4427975 miR-150 specific RT primer and PCR probe and 2 primers: https://www.thermofisher.com/order/genomedatabase/details/microrna/000473?CID=&ICID=&subtype= U6 specific RT primer and PCR probe and 2 primers: https://www.thermofisher.com/order/genomedatabase/details/microrna/001093?CID=&ICID=&subtype= ThermoFisher Real-Time PCR (qPCR) Learning Center: https://www.thermofisher.com/uk/en/home/life-science/pcr/real-time-pcr/real-time-pcr-learningcenter.html RNA extraction 1. Add 0.5 ml of TRIzol reagent to the 0.25ml of fraction you put aside for RNA extraction in week 1. 2. Incubate sample for 5 minutes at room temperature. Add 0.2 mL of chloroform per 1 mL of TRIzol used. Cap tube securely and shake vigorously by hand for 15 seconds. 3. Incubate sample for 3 minutes at room temperature. Centrifuge sample at 13,000 RPM for 15 minutes at room temperature. Remember to have the centrifuge balanced (possibly with other peoples sample(s)). The sample will separate into a pale green, organic phase, an interphase, and a colourless upper aqueous phase. 4. Transfer very carefully to another tube the phase that contains the RNA (you must find this information yourself if you are unsure which layer contains the RNA). Precipitate the RNA by mixing with cold isopropyl alcohol. Use 0.5 mL of isopropyl alcohol per 1 mL of TRIzol used. Incubate samples for 10 minutes at room temperature then centrifuge at 13,000 RPM for 10 minutes. Remember to have the centrifuge balanced. 5. Remove the supernatant and wash the pellet once with 75% ethanol, adding 1 mL of ethanol per 1 mL of TRIzol used. Vortex samples and centrifuge at 13,000 RPM for 5 minutes. Remember to have the centrifuge balanced. 6. Remove all ethanol, air-dry for 5 minutes, and then dissolve in 20 µl PCR water or DEPCtreated water by pipetting the solution up and down. Incubate for 10 minutes at 60°C if necessary. 7. Quantify your RNA using the nanodrop and record your values (you will need to know the concentration when preparing your reverse transcription reactions) 8. Ensure your tube is labelled with you name, fraction number and RNA. 9. Keep samples on ice. Reverse Transcription (RT) 1. You will be given 15 μl of the master mix. Before the session, read the RT instructions from the TaqMan Small RNA Assays Quick Reference Card to ensure you understand the contents of the master mix that has been prepared for you. 2. Add 7 μl of the master mix to 2 PCR tubes (smaller than eppendorfs). 3. Follow the rest of the TaqMan Small RNA Assays Quick Reference Card protocol from step 3 (note- you are preparing single-stranded RNA, so follow d.). In terms of which RT primer to add to each tube: 2 tubes should be split as follows: Tube 1- RNA prep + miR-150 RT Primer; Tube 2RNA prep + U6 snRNA RT Primer. Before the session, you will need to use your Nanodrop data to determine how much you have to dilute your RNA prep by (using DEPC-treated water) to have 1-10 ng total RNA in the reaction. 4. Place your clearly labelled PCR tubes (smaller than Eppendorf tubes) in the ice bucket next to the thermal cycler. 5. The thermal cycle will perform a run using the following conditions (will take about an hour): • Mode: Standard • Reaction volume: 15 µL • Thermal cycling conditions: Step Time Temperature Hold 30 minutes 16°C Hold 30 minutes 42°C Hold 5 minutes 85°C Hold ∞ 4°C Session 4 Flow cytometry analysis of HepG2 cells 1. Using the Cat 2 cabinet, remove media from the plate and add to 15 ml falcon tube. 2. Add 3 ml PBS to plate to wash cells, remove PBS and add to the media in the 15 ml falcon tube. 3. Add 0.5 ml trypsin to plate and put to 37°C for 5 minutes 4. Add 3 ml PBS, mix using the pipette and add cells/PBS to the 15 ml falcon tube 5. Spin 1,000 RPM for 5 minutes, pour off supernatant, add 5 ml PBS and re-suspend cell pellet, then spin again 1,000 RPM for 5 minutes 6. Pour off supernatant, add 5 ml PBS and re-suspend cell pellet, then spin again 1,000 RPM for 5 minutes 7. Resuspend after your final spin in 1 ml PBS and place sample in a labelled Eppendorf tube. 8. Take your sample to the BD Accuri flow cytometer and run your samples using count and FITC as your experimental axis, run 10 000 cells through for each run. 9. Control samples (no exosomes added) will similarly be analysed. 10. Save your data to a memory stick. Quantitative PCR (qPCR) amplification of RT reaction samples Before the session, read the Quantitative PCR (qPCR) amplification instructions from the TaqMan Small RNA Assays Quick Reference Card to see how to prepare required 4 tubes containing the reagents below: – TaqMan® Universal PCR Master Mix II, no UNG – Nuclease-free Water – TaqMan Small RNA Assay (20X) (1 probe and 2 primers for miR-150 OR 1 probe and 2 primers for U6 snRNA) – Product from RT reaction (one of the 4 tubes) 4 tubes will be set up for you containing 18.67 μl of a master mix containing all of the reagents except your product from the RT reaction: – Tube 1 miR-150 RT reaction with miR-150 Taqman Small RNA Assay – duplicate 1 – Tube 2 miR-150 RT reaction with miR-150 Taqman Small RNA Assay – duplicate 2 – Tube 3 U6 snRNA RT reaction with U6 snRNA Taqman Small RNA Assay – duplicate 1 – Tube 4 U6 snRNA RT reaction with U6 snRNA Taqman Small RNA Assay – duplicate 2 1. Follow the published instructions to add the correct RT reaction to each tube in the correct amount. 2. Take the samples to an academic who will add them to a qPCR plate. Note- no template controls (i.e. water added instead of RT reaction) will also be run on the plate for both the miR-150 Taqman Small RNA Assay and U6 snRNA Taqman Small RNA Assay reactions. 3. The qPCR will then be run as instructed in the “Set up and run the real–time PCR instrument” section of the general guide. 4. Results will be uploaded onto Aula. School of Life Sciences Assessment Brief Academic Year 2021-22 Section 1: Key information Module Code 7029BMS Module Name Genomic and regenerative medicine Semester 1 Status Normal Module Leader Steven Foster ([email protected]) Assessment Title CW2 Core /Applied Core Applied Core Credit weighting 10 credits Group/Individual Individual assessment Task outline You must produce a 2000-word laboratory book which is a suitable record of your activities in the laboratory, data analysis, critical analysis and reflections made over four laboratory sessions. Submission deadline/attendance date Labs are scheduled in weeks 9 to 10 – see your individual timetable for details. Report submission deadline is at the end of week 13 (Friday 15th April 2022 1800h) 24-hour grace period applies to allow for technical issues – submissions will be accepted up to 1800h on Saturday 16th April 2022. Submission/attendance instructions Due to the nature of the assessment, and the Learning Outcomes that are linked to this, it is a requirement that you attend on site laboratory classes in order to complete this assessment. The assessment requires you to plan, conduct, record and reflect on the results obtained from a series of laboratory practical sessions. If circumstances are going to prevent the onsite collection of data during semester 2, you must contact the module leader as soon as possible before the laboratory sessions to discuss the option of deferring both the laboratory work and coursework submission. Failure to attend any of the laboratory sessions without a suitable reason may mean you will not be permitted to submit the report and the coursework will be recorded as Absent (ABS). In these cases it is at the discretion of the Progression and Awards Board as to whether you will be permitted a resit attempt. Your report should be submitted via the Turnitin link on the 7029BMS Aula site. Do not include your name on your submitted work. Word or time limit 2000 words You should state your word count at the end of your work. If you exceed the word limit by more than 10% i.e., if you exceed 2200 words, then you will be penalised by deduction of 10% of your final mark. Work that is more than 30% above the allocated word limit (i.e., 2600 words or more) will only be read up to the allocated limit. Special instructions By attending the laboratories and submitting this assessment, you are declaring yourself fit to do so. If you are not fit to submit at this time you may apply for extension to the deadline or deferral to the next assessment period (see Extension and Deferral request instructions). Please note that if an extension to the deadline is granted, the 24 hour grace period DOES NOT apply. By submitting this assessment you agree to the following statement: I confirm that this CW submission represents my own work, and I have not received any unauthorised assistance. I understand the rules around plagiarism, collusion and contract cheating and that it is my responsibility to act with honesty and integrity in the assessment process. I understand that there will be no tolerance towards academic dishonesty, and that cheating can and will lead to serious consequences. Section 2- Detail of the Assessment task Students should produce a laboratory book using the layout provided below. Students should also consult the lab schedule for further information and hints and tips. LAYOUT TO USE FOR LABORATORY BOOK (Sub-headings that should be included in final laboratory book in bold; hints and tips that should be removed from final laboratory book in italics) Overall aim Given what is known about the macrophage-derived exosomes and their potential effects on cancer cells, outline what you think the overall aim(s) of the project was (i.e., can you suggest a hypothesis and/or scientific questions being tested). Findings from relevant related published studies should be used to introduce the project and support your statements. Session 1 Objective(s): summarise what the goal of each procedure was this session (i.e., say what you did and why). Reflection and planning for the next session: For example, you could discuss: which aspects of the session went well for you and why; if any aspects did not go well and what you would you do differently next time if had to repeat the same session; if what happened this week altered your plans for the following week; etc. Do not simply repeat the procedures you did in the lab. Procedures: Critically analyse the techniques/schedule/plan. Discuss if there are any modifications or additions you would make to the protocols and approaches to improve the experiment and potentially increase the likelihood of achieving the aims. Explain your reasoning. Do not waste words repeating the text from the lab schedule. Session 2 Objective(s): summarise what the goal of each procedure was this session (i.e., say what you did and why). Reflection and planning for the next session: For example, you could discuss: which aspects of the session went well for you and why; if any aspects did not go well and what you would you do differently next time if had to repeat the same session; if what happened this week altered your plans for the following week; etc. Do not simply repeat the procedures you did in the lab. Procedures: Critically analyse the techniques/schedule/plan. Discuss if there are any modifications or additions you would make to the protocols and approaches to improve the experiment and potentially increase the likelihood of achieving the aims. Explain your reasoning. Do not waste words repeating the text from the lab schedule. Session 3 Objective(s): summarise what the goal of each procedure was this session (i.e., say what you did and why). Reflection and planning for the next session: For example, you could discuss: which aspects of the session went well for you and why; if any aspects did not go well and what you would you do differently next time if had to repeat the same session; if what happened this week altered your plans for the following week; etc. Do not simply repeat the procedures you did in the lab. Procedures: Critically analyse the techniques/schedule/plan. Discuss if there are any modifications or additions you would make to the protocols and approaches to improve the experiment and potentially increase the likelihood of achieving the aims. Explain your reasoning. Do not waste words repeating the text from the lab schedule. Results: present your data in an appropriate format and provide accompanying text throughout briefly introducing the figures and describing the data (i.e. provide a narrative to follow). Data analysis: discuss what your data indicates and whether each of your goals were achieved. Offer possible explanations for any deviation from predicted results. Note- you will not be assessed on whether your experiments worked or not, you will be assessed on your critical analysis of what was expected and what the data indicates. Session 4 Objective(s): summarise what the goal of each procedure was this session (i.e., say what you did and why). Reflection and planning for the next session: For example, you could discuss: which aspects of the session went well for you and why; if any aspects did not go well and what you would you do differently next time if had to repeat the same session; if what happened this week altered your plans for the following week; etc. Do not simply repeat the procedures you did in the lab. Procedures: Critically analyse the techniques/schedule/plan. Discuss if there are any modifications or additions you would make to the protocols and approaches to improve the experiment and potentially increase the likelihood of achieving the aims. Explain your reasoning. Do not waste words repeating the text from the lab schedule. Results: present your data in an appropriate format and provide accompanying text throughout briefly introducing the figures and describing the data (i.e. provide a narrative to follow). Data analysis: discuss what your data indicates and whether each of your goals were achieved. Offer possible explanations for any deviation from predicted results. Note- you will not be assessed on whether your experiments worked or not, you will be assessed on your critical analysis of what was expected and what the data indicates. Overall discussion Discuss if the findings have helped address the overall aim/hypothesis/scientific question. Elaborate on how the findings fit (or not) with previously published studies and whether they could advance knowledge of the molecular mechanisms of liver cancer progression. Also comment on future experiments that could be performed to further confirm findings and/or move the project forward (to confirm already tested phenotypes using further assays and/or further experiments to test for additional phenotypes/effects) – think about what other experiments may need to be performed if this project was being prepared for publication. Detail of submission/ attendance instructions A DRAFT Turnitin link is available in the Course Community Aula site to allow you to check your similarity score prior to making your final submission. You may submit multiple times to this link, but do remember that obtaining a similarity report may take up to 24 hours. The FINAL Turnitin link on the module Aula page is for submission of your work for assessment. You may submit only ONCE to this link. Remember that submission make take some time to complete, so aim to submit several hours before the deadline. The TurnitinUK system will record the date and time of your submission and cannot be over-written. Please convert your final submission to a PDF format as these suffer less from formatting changes . If you experience any technical problems when trying to submit your work, please consult Aula help via the question mark link. If these problems are experienced at the time of the submission deadline and cannot be quickly resolved, please capture screenshots as evidence and email these and your completed assessment to the module leaders asap. Word count details The word limit for this assignment is 2000 words (+/-10%) The following are included in your word allowance: The text of your written work Reference citations and reference to Figures and Tables within the text The following are excluded from your word allowance: The title Figure/Table headings Figure/Table legends that contain information required for the figure or table (e.g. descriptions of different panels within the figure, or explanations of acronyms and abbreviations). However, long descriptive paragraphs used as legends ARE included in the word count (e.g., descriptions of data/results). Your name Student ID number, course, module name/code etc. Reference list The word count details Section 3: Help and Support The marking rubric and criteria is available later in this document. These will be used to help guide students, along with instructions and hints and tips provided within the lab schedule. Students will also have a regular opportunity to speak with the module leader during timetabled online sessions and weekly academic surgeries. If you have a special requirement such as a variation of assessment need please contact the disabilities team. Link to additional assessment information https://coventry.aula.education/?#/dashboard/3d0837cb-7c12-4f20-b493-a6d53b32b011/community/feed Section 4: Learning Outcomes and Marking Rubric Mapping to module This assessment is designed to assess Learning Outcomes 1 and 3 of the module: Learning outcomes 1. Critically review current knowledge and understanding of the molecular and cellular basis of tissue regeneration, selected inherited diseases and cancers. 3. Conduct, analyse, report and critically reflect on a series of laboratory experiments in the field of stem cell biology. Mapping to course Learning Outcomes This assessment relates to the following Course Learning Outcomes: MSc Biomedical Science: CS1: Critically analyse, evaluate and interpret knowledge and practice with regard to Biomedical Science. CS2: Collect, analyse and present data using appropriate methods. CS3: Apply scientific methods to the critical analysis of literature, reflection, and information searching in areas of Biomedical Science TS7: Innovative and problem-solving capabilities: the ability to apply transferable skills to the execution of individual and group projects involving the definition, analysis and resolution of complex problems. PS1: Undertake laboratory skills: the ability to work safely in the laboratory, undergoing progressively more advanced laboratory-based investigations based on competence in techniques appropriate to Biomedical Science PS2: Undertake Laboratory skills: Carry out appropriate measurements / audits aligned to the translation of information from basic to clinical science. PS3: Work effectively in a team: the ability to operate, to lead and collaborate in a team in order to solve problems of a practical (experimental) nature and to provide appropriate solutions. Task type/scheduling rationale This assessment task allows you to continue to further develop your laboratory and critical analysis skills following on from the 7027BMS skills module. The nature of the required critical analysis approach promotes independence and provides other skills often utilised in the research lab environment, adding further authenticity to the activities undertaken. This assessment deadline will provide the maximum possible time after the final session to complete the write-up and attend coursework support sessions. Indicative Marking Criteria The criteria detailed below will be used to assess your coursework at MSc level. You should read through the whole document carefully to gain an appreciation of the level of performance that is required to achieve each grade. This document should also be used alongside the provided coursework support documents and support sessions. In addition, you should also independently research the background related to your coursework and find other sources of material in addition to those provided by this module. Overall aim 5% High distinction ( 100 – 95 – 90 – 88 – 85 – 82) Distinction (78 – 75 – 72) Merit (68 – 65 – 62) High Pass (58 – 55 52) Low Pass (48 – 45 42) Fail (35 – 30 – 20 – 10 – 0) Overall aim or hypothesis of the set of experiments is excellently introduced and explained. Particularly easy to follow logic and arguments. Highly relevant literature used to support statements. The overall aim or hypothesis of the set of experiments is excellently introduced and explained. Very easy to follow logic and arguments. Very relevant literature used to support statements. The overall aim or hypothesis of the set of experiments is very well introduced and explained. Easy to follow logic and arguments. Mainly relevant literature used to support statements. The overall aim or hypothesis of the set of experiments is introduced and explained, though some explanations may be lacking or there may be some poorly expressed ideas. Quite easy to follow logic and arguments, though there may be some minor flaws in logic or understanding. Some relevant literature used to support statements. The overall aim or hypothesis of the set of experiments is reasonably well introduced and explained, though several explanations may be lacking or ideas may be poorly expressed. Difficult to follow logic and arguments in places, and there may be some flaws in logic or understanding. Literature used to support statements could be more directly relevant. The overall aim or hypothesis of the set of experiments is not introduced and explained. Explanations lack clarity or are very confused. Ideas are very poorly expressed. Section poorly constructed with some major flaws in logic or understanding. May be lacking use of relevant literature. Highly relevant and concise introduction to Very relevant and concise introduction to concepts that Relevant introduction Poor introduction to to concepts that concepts that underpin the work underpin the work Objective Very s and focussed and concise Very poor introduction to concepts that procedur es 25% introduction to concepts that underpin the work that week. Ration ale for methodology discussed, with very clear links between method and theory highlighted. Include cross referencing to methods that have been followed (e.g. course material/rese arch papers). Include a combination of sources, which are combined to construct academic level critical analysis. Full consideration of shortcomings and concepts that underpin the work that week. Rationale for methodology discussed, with very clear links between method and theory highlighted. Include cross referencing to methods that have been followed (e.g. course material/research papers). Include a combination of sources, which are combined to construct excellent critical analysis. Nearfull consideration of shortcomings and limitations of present design as well as excellent suggestions for improvements based on evidence. underpin the work that week, but contains minor omissions or misconceptions. Rati onale for methodology discussed, with good links between method and theory highlighted. Include cross referencing to methods that have been followed (e.g. course material/research papers). Include a combination of sources which are combined to construct a very good critical analysis of aspects. Some consideration of shortcomings and limitations of present design as well as very good suggestions for improvements based on evidence. that week, but may contain some omissions or misconceptions. Rati onale for methodology discussed, with some links between method and theory highlighted. Include some cross referencing to methods that have been followed (e.g. course material/research papers). Use different sources, which are combined to construct a good critical analysis of some aspects. Some consideration of shortcomings and limitations of present design, though there may be some omissions or flaws in understanding. Some suggestions for improvements largely based on evidence. that week, with several omissions or misconceptions. Rati onale for methodology poorly explained, with few links between method and theory highlighted. Poor cross referencing to methods that have been followed (e.g. course material/research papers). Critical analysis provided but lacks depth. There may be several errors. Consideration of shortcomings and limitations of present design may contain omissions or flaws in understanding. Suggestions for improvements may be brief or lack supporting evidence. underpin the work that week, with several major omissions or misconceptions. Rati onale for methodology very poorly explained, with no links between method and theory highlighted. Cross referencing to methods that have been followed (e.g. course material/research papers) may be lacking. No or minimal critical analysis. There may be numerous errors. Minimal or no consideration of shortcomings and limitations of present design or flaws in understanding. May be lacking consideration of possible improvements. limitations of present design as well as excellent suggestions for improvement s based on evidence. Results 10% Data presented in a highly appropriate format. Concise accompanyin g text provided throughout that very clearly introduces figures and describes the data. Has an particularly clear narrative that is very easy to follow and understand. Data presented in a very appropriate format. Concise accompanying text provided throughout that very clearly introduces figures and describes the data. Has a very clear narrative that is easy to follow and understand. Data presented in an appropriate format. Largely c…

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