The authors would like to thank Penn State Behrend and the Hamot Health Foundation for funding, as well as the Penn State Microscopy Facility in University Park, PA.

1. Preparation of buffers and reagents
NOTE: Make cell culture reagents in cell culture hood to maintain sterility.
<ol>
	<li>Preparation of cell culture reagents
	<ol>
		<li>Prepare normal high glucose DMEM by adding 50 mL of FBS&#160;and 5 mL of penicillin-streptococcus (Pen-Strep) into 500 mL of high glucose DMEM and store at 4 &#176;C. Use this media for culturing and expanding cells.</li>
		<li>Prepare exosome-depleted high glucose DMEM by adding 50 mL of exosome-deplet...

<ol>
	<li>Pan, B. T., Teng, K., Wu, C., Adam, M., Johnstone, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopic+evidence+for+externalization+of+the+transferrin+receptor+in+vesicular+form+in+sheep+reticulocytes.">Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes.</a> The Journal of Cell Biology. 101 (3), 942-948 (1985).</li><li>Harding, C., Heuser, J., Stahl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-mediated+endocytosis+of+transferrin+and+recycling+of+the+transferrin+receptor+in+rat+reticulocytes.">Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes.</a> The Journal of Cell Biology. 97 (2), 329-339 (1983).</li><li>Valadi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome-mediated+transfer+of+mRNAs+and+microRNAs+is+a+novel+mechanism+of+genetic+exchange+between+cells.">Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.</a> Nature Cell Biology. 9 (6), 654-659 (2007).</li><li>Raposo, G., Stoorvogel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles:+Exosomes,+microvesicles,+and+friends.">Extracellular vesicles: Exosomes, microvesicles, and friends.</a> The Journal of Cell Biology. 200 (4), 373-383 (2013).</li><li>Rontogianni, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+profiling+of+extracellular+vesicles+allows+for+human+breast+cancer+subtyping.">Proteomic profiling of extracellular vesicles allows for human breast cancer subtyping.</a> Communications Biology. 2 (1), 1-13 (2019).</li><li>Lane, R. E., Korbie, D., Hill, M. M., Trau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+as+circulating+cancer+biomarkers:+opportunities+and+challenges.">Extracellular vesicles as circulating cancer biomarkers: opportunities and challenges.</a> Clinical and Translational Medicine. 7 (1), 14 (2018).</li><li>Thompson, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+in+neurodegenerative+disease-pathogenesis+to+biomarkers.">Extracellular vesicles in neurodegenerative disease-pathogenesis to biomarkers.</a> Nature Reviews Neurology. 12 (6), 346-357 (2016).</li><li>O'Brien, K., Ughetto, S., Mahjoum, S., Nair, A. V., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake,+functionality,+and+re-release+of+extracellular+vesicle-encapsulated+cargo.">Uptake, functionality, and re-release of extracellular vesicle-encapsulated cargo.</a> Cell Reports. 39 (2), 110651 (2022).</li><li>Joshi, B. S., de Beer, M. A., Giepmans, B. N. G., Zuhorn, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis+of+extracellular+vesicles+and+release+of+their+cargo+from+endosomes.">Endocytosis of extracellular vesicles and release of their cargo from endosomes.</a> ACS Nano. 14 (4), 4444-4455 (2020).</li><li>Th&#233;ry, C., Amigorena, S., Raposo, G., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+exosomes+from+cell+culture+supernatants+and+biological+fluids.">Isolation and characterization of exosomes from cell culture supernatants and biological fluids.</a> Current Protocols in Cell Biology. 30 (1), 3-22 (2006).</li><li>Willms, E., Caba&#241;as, C., M&#228;ger, I., Wood, M. J. A., Vader, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicle+heterogeneity:+Subpopulations,+isolation+techniques,+and+diverse+functions+in+cancer+progression.">Extracellular vesicle heterogeneity: Subpopulations, isolation techniques, and diverse functions in cancer progression.</a> Frontiers in Immunology. 9, 738 (2018).</li><li>Tzaridis, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicle+separation+techniques+impact+results+from+human+blood+samples:+Considerations+for+diagnostic+applications.">Extracellular vesicle separation techniques impact results from human blood samples: Considerations for diagnostic applications.</a> International Journal of Molecular Sciences. 22 (17), 9211 (2021).</li><li>Liangsupree, T., Multia, E., Riekkola, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+isolation+and+separation+techniques+for+extracellular+vesicles.">Modern isolation and separation techniques for extracellular vesicles.</a> Journal of Chromatography A. 1636, 461773 (2021).</li><li>G&#225;mez-Valero, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-exclusion+chromatography-based+isolation+minimally+alters+extracellular+vesicles'+characteristics+compared+to+precipitating+agents.">Size-exclusion chromatography-based isolation minimally alters extracellular vesicles' characteristics compared to precipitating agents.</a> Scientific Reports. 6 (1), 1-9 (2016).</li><li>Mol, E. A., Goumans, M. J., Doevendans, P. A., Sluijter, J. P. G., Vader, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Higher+functionality+of+extracellular+vesicles+isolated+using+size-exclusion+chromatography+compared+to+ultracentrifugation.">Higher functionality of extracellular vesicles isolated using size-exclusion chromatography compared to ultracentrifugation.</a> Nanomedicine: Nanotechnology, Biology, and Medicine. 13 (6), 2061-2065 (2017).</li><li>Campos-Silva, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+detection+of+extracellular+vesicles+immune-captured+from+urine+by+conventional+flow+cytometry.">High sensitivity detection of extracellular vesicles immune-captured from urine by conventional flow cytometry.</a> Scientific Reports. 9 (1), 2042 (2019).</li><li>Norman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L1CAM+is+not+associated+with+extracellular+vesicles+in+human+cerebrospinal+fluid+or+plasma.">L1CAM is not associated with extracellular vesicles in human cerebrospinal fluid or plasma.</a> Nature methods. 18 (6), 631-634 (2021).</li><li>Th&#233;ry, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+information+for+studies+of+extracellular+vesicles+2018+(MISEV2018):+a+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines.">Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.</a> Journal of Extracellular Vesicles. 7 (1), 1535750 (2018).</li><li>Mathieu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificities+of+exosome+versus+small+ectosome+secretion+revealed+by+live+intracellular+tracking+of+CD63+and+CD9.">Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9.</a> Nature Communications. 12 (1), 1-18 (2021).</li><li>Guo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+simplified+dichotomic+size-exclusion+chromatography+for+isolating+extracellular+vesicles+toward+clinical+applications.">Establishment of a simplified dichotomic size-exclusion chromatography for isolating extracellular vesicles toward clinical applications.</a> Journal of Extracellular Vesicles. 10 (11), 12145 (2021).</li><li>Benedikter, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafiltration+combined+with+size+exclusion+chromatography+efficiently+isolates+extracellular+vesicles+from+cell+culture+media+for+compositional+and+functional+studies.">Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies.</a> Scientific Reports. 7 (1), 1-13 (2017).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> Journal of Visualized Experiments: JoVE. (44), e2359 (2010).</li><li>Grassucci, R. A., Taylor, D. J., Frank, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+macromolecular+complexes+for+cryo-electron+microscopy.">Preparation of macromolecular complexes for cryo-electron microscopy.</a> Nature Protocols. 2 (12), 3239 (2007).</li><li>Williams, R. L., Urb&#233;, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+shape+of+the+ESCRT+machinery.">The emerging shape of the ESCRT machinery.</a> Nature Reviews Molecular Cell Biology. 8 (5), 355-368 (2007).</li><li>Willms, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+release+subpopulations+of+exosomes+with+distinct+molecular+and+biological+properties.">Cells release subpopulations of exosomes with distinct molecular and biological properties.</a> Scientific Reports. 6 (1), 1-12 (2016).</li><li>Lehrich, B. M., Liang, Y., Fiandaca, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foetal+bovine+serum+influence+on+in+vitro+extracellular+vesicle+analyses.">Foetal bovine serum influence on in vitro extracellular vesicle analyses.</a> Journal of Extracellular Vesicles. 10 (3), 12061 (2021).</li><li>Kornilov, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+ultrafiltration-based+protocol+to+deplete+extracellular+vesicles+from+fetal+bovine+serum.">Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum.</a> Journal of Extracellular Vesicles. 7 (1), 1422674 (2018).</li><li>B&#246;ing, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-step+isolation+of+extracellular+vesicles+by+size-exclusion+chromatography.">Single-step isolation of extracellular vesicles by size-exclusion chromatography.</a> Journal of Extracellular Vesicles. 3 (1), (2014).</li><li>Zhang, X., Borg, E. G. F., Liaci, A. M., Vos, H. R., Stoorvogel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+three+step+protocol+to+isolate+extracellular+vesicles+from+plasma+or+cell+culture+medium+with+both+high+yield+and+purity.">A novel three step protocol to isolate extracellular vesicles from plasma or cell culture medium with both high yield and purity.</a> Journal of Extracellular Vesicles. 9 (1), 1791450 (2020).</li><li>Guerreiro, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+extracellular+vesicle+isolation+by+combining+cell+media+modifications,+ultrafiltration,+and+size-exclusion+chromatography.">Efficient extracellular vesicle isolation by combining cell media modifications, ultrafiltration, and size-exclusion chromatography.</a> PLoS ONE. 13 (9), 02024276 (2018).</li><li>On&#243;di, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+high-purity+extracellular+vesicles+by+the+combination+of+iodixanol+density+gradient+ultracentrifugation+and+bind-elute+chromatography+from+blood+plasma.">Isolation of high-purity extracellular vesicles by the combination of iodixanol density gradient ultracentrifugation and bind-elute chromatography from blood plasma.</a> Frontiers in Physiology. 9, 1479 (2018).</li><li>Yuana, Y., Levels, J., Grootemaat, A., Sturk, A., Nieuwland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-isolation+of+extracellular+vesicles+and+high-density+lipoproteins+using+density+gradient+ultracentrifugation.">Co-isolation of extracellular vesicles and high-density lipoproteins using density gradient ultracentrifugation.</a> Journal of Extracellular Vesicles. 3 (1), (2014).</li></ol>

SEC is a user-friendly method for adequately separating EVs from conditioned CCM. In order to specifically isolate cell derived EVs, careful consideration of the type of CCM and its supplements must be taken into account. Many cell culture medias need to be supplemented with FBS, which contains EVs derived from the animal in which the serum was harvested. These serum EVs may saturate and mask any signal produced by EVs derived from cells in culture26. Therefore, when performing experiments, EV-dep...

The explosion of interest in studying extracellular vesicles (EVs) has been accompanied by major advancements in the technologies and techniques used to separate and study these nano-sized, heterogeneous particles. In the time since their discovery nearly four decades ago1,2, these small membranous structures have been found to contain bioactive lipids, nucleic acids, and proteins, and play major roles in intercellular communication3,4. EVs are released from all cell types and are therefore present in all biological fluids, including blood plasma and serum, saliva, and urine. EVs within these fluids hold great promise to serve as non-invasive biomarkers for various diseases, including neuroinflammatory and neurodegenerative diseases, cancers, and autoimmune disorders5,6,7. Further, in vitro mechanistic studies can be performed through cell culture techniques by separating EVs released into the culture medium3,8,9.
To understand the role of EVs in disease pathophysiology, adequate separation from the fluid in which they are found is paramount. The gold standard for EV separation has long been differential ultracentrifugation (dUC)10, however more sophisticated techniques have arisen to achieve better separation of EVs from other extracellular components. Some of these techniques include density gradients, asymmetrical-flow field-flow fraction (A4F), flow cytometry, immunocapture, polyethylene glycol precipitation, and size exclusion chromatography (SEC)11,12,13. Each technique has its own set of advantages and disadvantages; however, SEC in particular has been shown to separate EVs from both biological fluids and cell culture supernatants quite effectively8,14,15. SEC also has the added bonus of being relatively straightforward and user-friendly.
SEC is a method that separates components of a fluid based on size. With this technique, a column of resin (either made in-house or purchased commercially) is used to fractionate a sample. Small particles in the sample get trapped between the beads within the resin, while larger particles are able to pass through the resin more freely, and thus elute earlier in the process. Because EVs are larger in size than many extracellular proteins and protein aggregates, EVs pass through the column faster and elute in earlier fractions than extracellular proteins14.
In this methods paper, the use of SEC for separation of EVs from cell culture media (CCM) from human oligodendrocytes under both control and endoplasmic reticulum (ER) stress conditions is outlined. Using this protocol, it is shown that EVs separated with this technique are found within specific fractions that can be pooled together and concentrated for downstream characterization, and that the separated EVs are derived from cells and not from an exogenous source such as fetal bovine serum (FBS) used to supplement the CCM. The presence of the canonical EV markers, CD63, CD81, and CD916,17,18,19 in the EV fractions, and their absence in the protein fractions is demonstrated with western blotting. Using transmission electron microscopy (TEM), EVs are visualized and display the expected morphology and are only observed in the EV fraction. Particles are also counted in the EV and protein fractions of both control and ER stress conditions, and a large number of particles within the expected size range of 50-200 nm in diameter are observed in the EV samples. Together, these data support the notion that SEC is an efficient and effective method for separating EVs from cell culture media.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>VWR</td>
			<td>97064-588</td>
			<td></td>
		</tr>
		<tr>
			<td>4X Laemmli Sample Buffer</td>
			<td>BioRad</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Unit, Ultracel, 3 KDa, 15mL</td>
			<td>Sigma-Aldrich</td>
			<td>UFC900308</td>
			<td>3 kDa cutoff</td>
		</tr>
		<tr>
			<td>Amicon Ultra-2 Centrifugal Filter Unit with Ultracel-3 membrane</td>
			<td>Sigma-Aldrich</td>
			<td>UFC200324</td>
			<td>3 kDa cutoff</td>
		</tr>
		<tr>
			<td>Ammonium Persulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A3678-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti rabbit IgG, HRP linked Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>7074V</td>
			<td>1:1000 Dilution</td>
		</tr>
		<tr>
			<td>Anti-Calnexin antibody</td>
			<td>Abcam</td>
			<td>&#160;ab22595</td>
			<td>1:500 Dilution</td>
		</tr>
		<tr>
			<td>Anti-CD9 Mouse Monoclonal Antibody</td>
			<td>BioLegend</td>
			<td>312102</td>
			<td>1:500 Dilution</td>
		</tr>
		<tr>
			<td>Anti-GM130 antibody [EP892Y] - cis-Golgi Marker</td>
			<td>Abcam</td>
			<td>ab52649</td>
			<td>1:500 Dilution</td>
		</tr>
		<tr>
			<td>Anti-mouse IgG, HRP-linked Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>7076V</td>
			<td>1:1000 Dilution</td>
		</tr>
		<tr>
			<td>Automatic Fraction Collector</td>
			<td>Izon Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BCA assay Kit</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Gatan Orius SC200</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cd63 Mouse anti Human</td>
			<td>BD</td>
			<td>556019</td>
			<td>1:1000 Dilution</td>
		</tr>
		<tr>
			<td>CD81 Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-23962</td>
			<td>1:1000 Dilution</td>
		</tr>
		<tr>
			<td>Cellstar Filter Cap Cell Culture Flasks</td>
			<td>Greiner Bio-One</td>
			<td>660175</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc MP Imager</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clarity Western ECL Substrate</td>
			<td>BioRad</td>
			<td>1705061</td>
			<td></td>
		</tr>
		<tr>
			<td>deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>dithiothreitol</td>
			<td>Sigma</td>
			<td>3483-12-3</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/High glucose with L-glutamine; without sodium</td>
			<td>Cytiva</td>
			<td>SH300022.FS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum Premium grade</td>
			<td>VWR</td>
			<td>97068-085</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, exosome-depleted</td>
			<td>Thermo Scientific</td>
			<td>A2720801</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>BioRad</td>
			<td>1610718</td>
			<td></td>
		</tr>
		<tr>
			<td>Great Value Nonfat Dry Milk</td>
			<td>Amazon</td>
			<td>B076NRD2TZ</td>
			<td></td>
		</tr>
		<tr>
			<td>HOG Human Oligodendroglioma Cell Line</td>
			<td>Sigma-Aldrich</td>
			<td>SCC163</td>
			<td></td>
		</tr>
		<tr>
			<td>Izon Science Usa Ltd qev Size Exclusion Columns 5pk</td>
			<td>Izon Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol &#62;99.8% ACS</td>
			<td>VWR</td>
			<td>BDH1135-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Glass plates</td>
			<td>BioRad</td>
			<td>1653310</td>
			<td>with 0.75mm spacers</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Short plates</td>
			<td>BioRad</td>
			<td>1653308</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Sigma-Aldrich</td>
			<td>492016</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin,Solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4458-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline PBS</td>
			<td>Fisher Scientific</td>
			<td>BP66150</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kits and Reagents</td>
			<td>Thermo Fisher Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce PVDF Transfer Membranes</td>
			<td>Thermo Scientific</td>
			<td>88518</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Western Blotting Filter Paper</td>
			<td>Thermo Scientific</td>
			<td>84783</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene-20 (TWEEN 20), 500mL</td>
			<td>Bio Basic</td>
			<td>TB0560</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease/phosphatase Inhibitor Cocktail (100X)</td>
			<td>Cell Signaling Technology</td>
			<td>5872S</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Anti-TSG101 antibody [EPR7130(B)]</td>
			<td>ABCam</td>
			<td>ab125011</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Slodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>SX0603</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Fisher Scientific</td>
			<td>BP922I-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate,&#8805;99.0% (GC), dust-free pellets</td>
			<td>Sigma-Aldrich</td>
			<td>75746-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine</td>
			<td>Sigma-Aldrich</td>
			<td>T9281-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TGX Stain-Free FastCast Acrylamide Kit, 10%</td>
			<td>BioRad</td>
			<td>1610183</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>FEI Tecnai 12 Biotwin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>BioRad</td>
			<td>1610716</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.25% protease with porcine trypsin, HBSS, EDTA; without calcium, magnesium</td>
			<td>Cytiva</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Tunicamycin</td>
			<td>Tocris</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeta View software</td>
			<td>Analytik</td>
			<td></td>
			<td>NTA software</td>
		</tr>
	</tbody>
</table>

size exclusion chromatography for separating extracellular vesicles from conditioned cell culture media

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain proteins, nucleic acids, and lipids that are reflective of the parent cell from which they are derived. Proper separation of EVs from other components in a sample allows for characterization of their associated cargo and lends insight into their potential as intercellular communicators and non-invasive biomarkers for numerous diseases. In the current study, oligodendrocyte derived EVs were isolated from cell culture media using a combination of state-of-the-art techniques, including ultrafiltration and size exclusion chromatography (SEC) to separate EVs from other extracellular proteins and protein complexes. Using commercially available SEC columns, EVs were separated from extracellular proteins released from human oligodendroglioma cells under both control and endoplasmic reticulum (ER) stress conditions. The canonical EV markers CD9, CD63, and CD81 were observed in fractions 1-4, but not in fractions 5-8. GM130, a protein of the Golgi apparatus, and calnexin, an integral protein of the ER, were used as negative EV markers, and were not observed in any fraction. Further, when pooling and concentrating fractions 1-4 as the EV fraction, and fractions 5-8 as the protein fraction, expression of CD63, CD81, and CD9 in the EV fraction was observed. The expression of GM130 or calnexin was not observed in either of the fraction types. The pooled fractions from both control and ER stress conditions were visualized with transmission electron microscopy and vesicles were observed in the EV fractions, but not in the protein fractions. Particles in the EV and protein fractions from both conditions were also quantified with nanoparticle tracking analysis. Together, these data demonstrate that SEC is an effective method for separating EVs from conditioned cell culture media.

Western blotting reveals adequate separation of EVs from CCM 
To evaluate the effectiveness of SEC for separating EVs from cell culture media, a western blot was run using each individual fraction from the control samples to probe expression of the three canonical EV markers, CD9, CD63 and CD81, as well as GM130 and calnexin18, which were used as negative controls (Figure 3). Albumin expression18 was also probed to ensure ...

Watch this Scientific Journal Video about Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media at JoVE.com

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain ...

Separation of extracellular vesicles from fluids like condition cell culture media can allow researchers to characterize vesticular cargo and explore their intracellular communication mechanisms. This technique allows for sufficient separation of EVs and extracellular proteins from conditioned cell culture media that is straightforward and user-friendly. To begin place PBS in a 37 degrees celsius water bath.Observe the control and tunicamycin treated cell culture flasks under a compound microscope with a 10 x and 100 x objective lens. Take notes of any differences between the flasks. Remove the medium from the flask and place it in a 50 milliliter tube.Then centrifuge the tube at 500 G for five minutes at four degrees celsius and transfer the super natant to a new tube. Centrifuge the tube at 2000 G for 20 minutes at four degrees celsius. Transfer the super natant to a new tube and place the tube on ice.Take four 15 milliliters three kilodaltons cutoff ultra filtration units and add five milliliters of PBS to each tube. Prime the filters by centrifuging the units at 4, 000 G for 10 minutes at four degrees celsius. After removing PBS from the units, divide supernatant from control and treated conditions into two ultra filtration units each with approximately 12.5 milliliters of media in each.Centrifuge the tubes at 4, 000 G for one hour 45 minutes at four degrees celsius or until the concentrated media or retentate in each unit is concentrated to 250 microliters or less. Transfer the retentate from both control units into a labeled 1.5 milliliter micro centrifuge tube and from treated units into another tube. Measure the total volume of the retentate in each micro centrifuge tube.If the volume is less than 500 microliters, add 0.22 micron filtered PBS until the final volume is 500 microliters. Place tubes in a minus 80 degree celsius freezer. Turn on the automatic fraction collector or AFC.Remove the column from four degrees celsius and allow the column to warm up to room temperature. Remove retentate samples from the minus 80 degrees celsius freezer and place them on ice to thaw, while waiting, set eight labeled 1.5 milliliter tubes in the AFC carousel with lids facing inwards. Insert the column into the AFC with the barcode facing the reader and adjust settings to collect eight fractions of 500 microliters each and buffer volume to default.Then verify the on-screen instructions to start the collection. After the storage buffer is completely absorbed into the top portion of the column matrix. Begin flushing the column by adding 15 milliliters of PBS to the column.Do not add PBS too early as it will dilute the storage buffer and prevent adequate flushing. Just before all 15 milliliters of PBS get absorbed by the matrix click okay to stop the flushing and remove any residual PBS from the top of the column matrix with a micro pipette. Do not touch the matrix.Add 500 microliters of sample to the center of the column and click okay to begin the run. After the sample is fully absorbed into the matrix immediately add eight milliliters of PBS to the column. AFC automatically dispels the void volume into the center of the carousel before collecting all eight fractions of 500 microliters each.After the run, remove fractions from the carousel and place them on ice. Then remove the void volume from the center of the carousel. Next, add 10 milliliters of PBS to the column to wash any residual sample from the column.And once the repented sample has been fully flushed add 15 milliliters of PBS to the column. As the final PBS is being absorbed by the matrix add 500 microliters of 0.5 molar sodium hydroxide to the center of the column's matrix. Then as the sodium hydroxide is absorbed, add 30 milliliters of PBS to the column and let it flush through.Once done with all samples preserve the column for later use by adding 15 milliliters of 0.05%sodium azide to the column. Leave approximately five milliliters of sodium azide on the top of the column matrix. Remove the column from the AFC and attach the top and bottom caps before placing the column at four degrees celsius for storage.Construct the ultra filtration unit. Obtain two milliliters, three kilodalton cutoff ultra filtration units per sample. Each unit is comprised of three parts, cone, filter and flow through cylinder.Label each part properly. Add one milliliter PBS to the filter portion and cap with the cone piece. Centrifuge the ultrafiltration unit cone side up at 3, 500 G for 10 minutes at four degrees celsius and remove the filtered PBS from the flow through cylinder.Flip the ultrafiltration unit upside down and centrifuge for two minutes at 1000 G at four degrees celsius to remove any remaining PBS. Combine four 500 microliters of each fraction and add them to the ultrafiltration unit. Then centrifuge columns coincide up for one hour, 45 minutes at 3, 500 G at four degrees celsius or until the retentate level is at 100 microliters.Remove the flow through cylinder and discard the flow through. Flip the ultra filtration unit upside down and centrifuge for two minutes at 1000 XG at four degrees celsius. Transfer the retentate in the cone to a 1.5 milliliter tube and make the sample volume to 150 microliters with 0.22 micron filtered PBS before placing the tubes in minus 80 degrees celsius freezer.A representative western block of individual fractions showed strong expression of CD9, CD63, and CD81. Infractions one to four with little or no expression infractions five to eight. No expression of GM130 or calnexin was observed in any fraction.Albumin was only present in fractions six to eight. The effectiveness of concentrating the EV and protein fractions was also evaluated. Expression of CD9 and CD63 was observed in the cell lysates.All three CD proteins were present in EV fractions of control and tunicamycin treated samples but not in the protein fractions. The tumor susceptibility gene 101 was present in cell lysates, but not in EV or protein fractions. The GM130 and calnexin were only observed in the cell lysates.In the western blot of exosome depleted cell culture media, the EV markers were present in the cell lysates but not in the media samples. Albumin expression was observed in the protein fractions and minimal expression in the cell lysates. TEM observations demonstrated spherical structures in the EV fractions of control and tunicamycin treated samples.These structures were not observed in either sample type protein fractions. Nanoparticle tracking analysis reveals differences in particle concentrations of control and treated fractions in EV fractions compared to the control, slightly more particles were present in the tunicamycin treatment. However, in the protein fractions, fewer particles were detected relative to EVs.It's important to have the same sample volume for the control and treated conditions, allowing for easy comparison of EVs from the different treatments. After separating EVs from the condition cell culture media we can characterize the protein, RNA and lipid content of the vesicles with proteomics RNA sequencing and lipidomics.

size-exclusion-chromatography-for-separating-extracellular-vesicles

Research

JoVE Journal

Neuroscience

3.2K Views.  Penn State Erie, The Behrend College. Separation of extracellular vesicles from fluids like condition cell culture media can allow researchers to characterize vesticular cargo and explore their intracellular communication mechanisms. This technique allows for sufficient separation of EVs and extracellular proteins from conditioned cell culture media that is straightforward and user-friendly. To begin place PBS in a 37 degrees celsius water bath.Observe the control and tunicamycin treated cell culture flasks under a compoun...

Video: Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

flow cytometric analysis of extracellular vesicles from cell-conditioned media

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry...

a size exclusion chromatography technique to purify mycobacterial extracellular vesicles

This video demonstrates the size exclusion chromatography (SEC) technique for purifying bacterial extracellular vesicles (EVs) from the culture filtrate of Mycobacterium tuberculosis (Mtb). The technique utilizes a porous resin-packed column to obtain purified fractions of EVs. The porous particles selectively allow smaller proteins to enter the pores, resulting in slower elution. At the same time, EVs are excluded from entering the pores due to their larger size and are eluted...

A Size Exclusion Chromatography Technique to Purify Mycobacterial Extracellular Vesicles

Remove the caps from the equilibrated SEC column, and slide the SEC column into the appropriate column mount. Carefully install it on the AFC tower. Ensure that the radio frequency identification tag on the column faces the AFC, and check that the connection between the column and the valve is secure. Place the waste outlet tubing in a collection container. From the SETUP screen, select Collection Schedule, and set the count to 13, and the size to 0.5 milliliters by pressing the minus or plus...

sec-based isolation of microglia-derived extracellular vesicles: a technique to isolate extracellular vesicles from microglia conditioned culture media

This video describes a method based on size-exclusion chromatography (SEC) to isolate extracellular vesicles or EVs derived from the brain immune cells&#8213;the microglia. The EVs act as molecular cargo to deliver mediators, which regulate an immune response. Therefore, the isolated EVs can be used as an in vitro model to study the microglia-neuron crosstalk during a neuronal immune...

SEC-based Isolation of Microglia-derived Extracellular Vesicles: A Technique to Isolate Extracellular Vesicles from Microglia Conditioned Culture Media

mycobacterium tuberculosis extracellular vesicle enrichment through size exclusion chromatography

Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography

This protocol describes size exclusion chromatography, a facile and reproducible technique for enriching Mycobacterium tuberculosis extracellular vesicles from culture supernatants.

Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography

Then, transfer the supernatant into a 10.4-milliliter polycarbonate tube. Place the tube into a 70.1 Ti rotor and ultracentrifuge it at 100,000 x g for 90 minutes at 4 degrees Celsius to pellet the EVs. After centrifugation, discard the supernatant and resuspend the pellet in 200 microliters of 0.2-micrometer filtered...

size exclusion chromatography based separation of bacterial extracellular vesicles: a technique to separate heterogenous population of gram-negative bacterial outer membrane vesicles based on their size

In this video, we demonstrate a technique&#8211;size exclusion chromatography&#8211;to separate a heterogenous population of outer membrane vesicles or OMVs derived from bacteria. These vesicles are nanosized spherical structures and are separated into large and small subpopulations based on their size through Size Exclusion...

Size Exclusion Chromatography Based Separation of Bacterial Extracellular Vesicles: A Technique to Separate Heterogenous Population of Gram-Negative Bacterial Outer Membrane Vesicles Based on Their Size

Mount the column on a ring stand in the vertical position, and fill the column with elution buffer to wet the walls, before draining the buffer until only about 1 centimeter remains. Then, carefully pipette the gel beads into the column, while simultaneously draining the excess buffer to prevent the beads from settling, until the column is packed to a height of approximately 2 centimeters below the bottom of the column...

size exclusion chromatography to analyze bacterial outer membrane vesicle heterogeneity

Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

Bacterial vesicles play important roles in pathogenesis and have promising biotechnological applications. The heterogeneity of vesicles complicates analysis and use; therefore, a simple, reproducible method to separate varying sizes of vesicles is necessary. Here, we demonstrate the use of size exclusion chromatography to separate heterogeneous vesicles produced by Aggregatibacter...

scalable biomanufacturing workflow to produce and isolate natural killer cell-derived extracellular vesicle-based cancer biotherapeutics

Author Spotlight: Scalable Isolation and Production of Therapeutic Immune Cell-Derived Vesicles for Solid Tumor Treatment

Natural killer cell-derived extracellular vesicles (NK-EVs) hold promising potential as cancer biotherapeutics. This methodology-based study presents a scalable closed-loop biomanufacturing workflow designed to continuously produce and isolate large quantities of high-purity NK-EVs. In-process control testing is performed throughout the biomanufacturing workflow, ensuring the EVs meet quality standards for product release.

Biomanufacturing and Characterization of NK-Extracellular Vesicles

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size-exclusion chromatography with multi-angle light scattering for protein oligomers

This video describes SEC-MALS, which leverages the abilities of size-exclusion chromatography (SEC) and multi-angle light scattering (MALS). In SEC-MALS, the SEC separates proteins or protein complexes based on their size, and the MALS detector determines the average molecular weight by measuring the intensity of the light scattered by the molecule at different...

Size-Exclusion Chromatography with Multi-Angle Light Scattering for Protein Oligomers

rapid assessment of membrane protein quality by fluorescent size exclusion chromatography

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

The present protocol describes a procedure to perform fluorescent size exclusion chromatography (FSEC) on membrane proteins to assess their quality for downstream functional and structural analysis. Representative FSEC results collected for several G-protein coupled receptors (GPCRs) under detergent-solubilized and detergent-free conditions are...

protein digestion, ultrafiltration, and size exclusion chromatography to optimize the isolation of exosomes from human blood plasma and serum

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic...

Filter the first 50 to 100 milliliters of buffer to a waste bottle in order to eliminate particulates from the dry filters. Then, filter the remainder to a clean, sterile bottle that has been previously washed with filtered, deionized water and kept to prevent dust from...

online size-exclusion and ion-exchange chromatography on a saxs beamline

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

The determination of the solution structure of a protein by small angle X-ray scattering (SAXS) requires monodisperse samples. Here, we present two possibilities to ensure minimal delays between sample preparation and data acquisition: online size-exclusion chromatography (SEC) and online ion-exchange chromatography...

extracting extracellular vesicles (evs) from adipose-derived mesenchymal stem cell culture

Extracting Extracellular Vesicles (EVs) from Adipose-Derived Mesenchymal Stem Cell Culture

nanoparticle tracking analysis for the quantification and size determination of extracellular vesicles

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

purification and characterization of extracellular vesicles from human adipose-derived mesenchymal stem cells

Author Spotlight: Standardizing and Improving the Extraction and Purification of Extracellular Vesicles from Human ADSCs

This protocol describes all procedures, from culturing human adipose-derived mesenchymal stem cells (ADSCs) and collecting supernatant to extracting extracellular vesicles (EVs) using...

Ultracentrifuge-Based Isolation of Extracellular Vesicles from Human ADSCs

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a novel culture model for human pluripotent stem cell propagation on gelatin in placenta-conditioned media

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic...

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Preparation of a High-quality Primary Cell Culture from Fish Pituitaries

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled environment. Despite vast differences in the physiology between mammals and fish, primary cell culture protocols from fish are often based on mammalian culture conditions, often with only minor modifications. The environmental differences affect not only body temperature, but also blood serum parameters such as osmolality, pH, and pH buffer capacity. As cell culture media and similar working solutions are meant to mimic characteristics of the extracellular fluid and/or blood serum to which a cell is adapted, it is crucial that these parameters are adjusted specifically to the animal in question.
The current protocol describes optimized primary culture conditions for medaka (Oryzias latipes). The protocol provides detailed steps on how to isolate and maintain healthy dissociated pituitary cells for more than one week and includes the following steps: 1. the adjustment of the osmolality to the values found in medaka blood plasma, 2. the adjustment of the incubation temperature to normal medaka temperature (here in the aquarium facility), and 3. the adjustment of the pH and bicarbonate buffer to values comparable to other fish species living at similar temperatures. The results presented using the described protocol promote physiologically meaningful results for medaka and can be used as a reference guide by scientists making primary cell cultures from other non-mammalian species.

Here we describe a protocol to prepare and maintain primary pituitary cell cultures from medaka (Oryzias latipes). The optimized conditions in this protocol take important parameters such as temperature, osmolality, and pH into consideration by mimicking the physiological conditions of the fish, thereby enabling physiologically more meaningful results.

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled ...

Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident immune cells in the brain, and sometimes the infiltrating bone marrow-derived macrophages (BMDMs), regulate the inflammatory profile of their microenvironment in the CNS. It is now accepted that the extracellular vesicle (EV) populations from immune cells act as immune mediators. Thus, their collection and isolation are important to identify their contents but also evaluate their biological effects on recipient cells. The present data highlight chronological requirements for EV isolation from microglia cells or blood macrophages including the ultracentrifugation and size-exclusion chromatography (SEC) steps. A non-targeted proteomic analysis permitted the validation of protein signatures as EV markers and characterized the biologically active EV contents. Microglia-derived EVs were also functionally used on primary culture of neurons to assess their importance as immune mediators in the neurite outgrowth. The results showed that microglia-derived EVs contribute to facilitate the neurite outgrowth in vitro. In parallel, blood macrophage-derived EVs were functionally used as immune mediators in spheroid cultures of C6 glioma cells, the results showing that these EVs control the glioma cell invasion in vitro. This report highlights the possibility to evaluate the EV-mediated immune cell functions but also understand the molecular bases of such a communication. This deciphering could promote the use of natural vesicles and/or the in vitro preparation of therapeutic vesicles in order to mimic immune properties in the microenvironment of CNS pathologies.

The present report highlights chronological requirements for extracellular vesicle (EV) isolation from microglia or blood macrophages. Microglia-derived EVs were evaluated as regulators of the neurite outgrowth while blood macrophage-derived EVs were studied in the control of C6 glioma cell invasion in in vitro assays. The goal is to better understand these EV functions as immune mediators in specific microenvironments.

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Post-Movie Subliminal Measurement (PMSM), for Investigating Implicit Social Bias

New knowledge is continuously gained from a social environment that can influence how people respond to each other. Such responses often occur implicitly, at a subliminal perceptual level, and related brain mechanisms can be experimentally isolated by presenting the stimuli quickly. Subliminal presentation of faces that belong to different ethnicity groups, races, or gender has been shown to be successful in investigating social implicit responses. However, many implicit responses are based on knowledge previously gained about the faces (e.g., sexual orientation, political views, and socioeconomic status) and not solely on physical appearance. Here, a novel method called post-movie subliminal measurement (PMSM) is presented. When watching a socially engaging movie, a spectator gains knowledge about the protagonist and becomes familiar with his/her identity and world views. When the face of the protagonist is presented subliminally after the movie, it evokes an implicit neural response depending on what is learned about the protagonist. With a vast number of movies available, each depicting a variety of people with different identities, the PMSM method enables investigation of the brain&#39;s complex implicit biases in a manner that resembles real-life social perceptions.

Post-Movie Subliminal Measurement PMSM, for Investigating Implicit Social Bias

This protocol describes the use of movies to investigate brain mechanisms underlying implicit social biases during functional magnetic resonance imaging. When the face of a protagonist is presented after a movie subliminally, it evokes an implicit response based on knowledge of the protagonist gained during the movie.

New knowledge is continuously gained from a social environment that can influence how people respond to each other. Such responses often occur implicitly, ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...