This work was partially supported by a Fund-A-Fellow grant from the Myotonic Dystrophy Foundation (MDF-FF-2014-0013) and the Scientist Development Grant from the American Heart Association (16SDG29320001).

Wildtype and Nestin-GFP transgenic mice were housed in the animal facility at the University of Minnesota. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Minnesota and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
1. Muscle Dissection and Single-cell Isolation
<ol>
	<li>Euthanize adult mice (6-10 weeks, both male and female) with tribromoethanol (250 mg/kg, i.p.) and sterilize their abdomen...

<ol>
	<li>Rennert, R. C., Sorkin, M., Garg, R. K., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+recruitment+after+injury:+lessons+for+regenerative+medicine.">Stem cell recruitment after injury: lessons for regenerative medicine.</a> Regenerative medicine. 7 (6), 833-850 (2012).</li><li>Sambasivan, 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=Pax7-expressing+satellite+cells+are+indispensable+for+adult+skeletal+muscle+regeneration.">Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration.</a> Development. 138 (17), 3647-3656 (2011).</li><li>Relaix, F., Zammit, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cells+are+essential+for+skeletal+muscle+regeneration:+the+cell+on+the+edge+returns+centre+stage.">Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage.</a> Development. 139 (16), 2845-2856 (2012).</li><li>von Maltzahn, J., Jones, A. E., Parks, R. J., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax7+is+critical+for+the+normal+function+of+satellite+cells+in+adult+skeletal+muscle.">Pax7 is critical for the normal function of satellite cells in adult skeletal muscle.</a> Proc Natl Acad Sci U S A. 110 (41), 16474-16479 (2013).</li><li>Lepper, C., Partridge, T. A., Fan, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+absolute+requirement+for+Pax7-positive+satellite+cells+in+acute+injury-induced+skeletal+muscle+regeneration.">An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration.</a> Development. 138 (17), 3639-3646 (2011).</li><li>Kuang, S., Charge, S. B., Seale, P., Huh, M., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+for+Pax7+and+Pax3+in+adult+regenerative+myogenesis.">Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis.</a> J Cell Biol. 172 (1), 103-113 (2006).</li><li>Murphy, M. M., Lawson, J. A., Mathew, S. J., Hutcheson, D. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+specification+of+side+population+cells+in+skeletal+muscle.">Myogenic specification of side population cells in skeletal muscle.</a> J Cell Biol. 159 (1), 123-134 (2002).</li><li>Tamaki, 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=Skeletal+muscle-derived+CD34+/45-+and+CD34-/45-+stem+cells+are+situated+hierarchically+upstream+of+Pax7++cells.">Skeletal muscle-derived CD34+/45- and CD34-/45- stem cells are situated hierarchically upstream of Pax7+ cells.</a> Stem Cells Dev. 17 (4), 653-667 (2008).</li><li>Dellavalle, 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=Pericytes+resident+in+postnatal+skeletal+muscle+differentiate+into+muscle+fibres+and+generate+satellite+cells.">Pericytes resident in postnatal skeletal muscle differentiate into muscle fibres and generate satellite cells.</a> Nat Commun. 2, 499 (2011).</li><li>Mitchell, K. 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=Identification+and+characterization+of+a+non-satellite+cell+muscle+resident+progenitor+during+postnatal+development.">Identification and characterization of a non-satellite cell muscle resident progenitor during postnatal development.</a> Nat Cell Biol. 12 (3), 257-266 (2010).</li><li>Pannerec, A., Formicola, L., Besson, V., Marazzi, G., Sassoon, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+skeletal+muscle+resident+progenitors+and+their+cell+fate+potentials.">Defining skeletal muscle resident progenitors and their cell fate potentials.</a> Development. 140 (14), 2879-2891 (2013).</li><li>Dellavalle, 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=Pericytes+of+human+skeletal+muscle+are+myogenic+precursors+distinct+from+satellite+cells.">Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells.</a> Nat Cell Biol. 9 (3), 255-267 (2007).</li><li>Kostallari, 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=Pericytes+in+the+myovascular+niche+promote+post-natal+myofiber+growth+and+satellite+cell+quiescence.">Pericytes in the myovascular niche promote post-natal myofiber growth and satellite cell quiescence.</a> Development. 142 (7), 1242-1253 (2015).</li><li>Birbrair, 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=Role+of+pericytes+in+skeletal+muscle+regeneration+and+fat+accumulation.">Role of pericytes in skeletal muscle regeneration and fat accumulation.</a> Stem Cells Dev. 22 (16), 2298-2314 (2013).</li><li>Yao, Y., Norris, E. H., Mason, C. E., Strickland, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+regulates+PDGFRbeta(+)+cell+stemness+and+muscle+development.">Laminin regulates PDGFRbeta(+) cell stemness and muscle development.</a> Nat Commun. 7, 11415 (2016).</li><li>Armulik, A., Genove, G., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes:+developmental,+physiological,+and+pathological+perspectives,+problems,+and+promises.">Pericytes: developmental, physiological, and pathological perspectives, problems, and promises.</a> Dev Cell. 21 (2), 193-215 (2011).</li><li>Dore-Duffy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes:+pluripotent+cells+of+the+blood+brain+barrier.">Pericytes: pluripotent cells of the blood brain barrier.</a> Curr Pharm Des. 14 (16), 1581-1593 (2008).</li><li>Dore-Duffy, P., Katychev, A., Wang, X., Van Buren, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+microvascular+pericytes+exhibit+multipotential+stem+cell+activity.">CNS microvascular pericytes exhibit multipotential stem cell activity.</a> J Cereb Blood Flow Metab. 26 (5), 613-624 (2006).</li><li>Birbrair, 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=Skeletal+muscle+pericyte+subtypes+differ+in+their+differentiation+potential.">Skeletal muscle pericyte subtypes differ in their differentiation potential.</a> Stem Cell Res. 10 (1), 67-84 (2013).</li><li>Gautam, J., Nirwane, A., Yao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+differentially+regulates+the+stemness+of+type+I+and+type+II+pericytes.">Laminin differentially regulates the stemness of type I and type II pericytes.</a> Stem Cell Research &amp; Therapy. 8 (1), 28 (2017).</li><li>Birbrair, 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=Pericytes:+multitasking+cells+in+the+regeneration+of+injured,+diseased,+and+aged+skeletal+muscle.">Pericytes: multitasking cells in the regeneration of injured, diseased, and aged skeletal muscle.</a> Front Aging Neurosci. 6, 245 (2014).</li></ol>

All authors have no conflict of interest to disclose.

Pericytes are multipotent perivascular cells22,23 located on the abluminal surface of capillaries21,26. In skeletal muscles, pericytes are able to differentiate along the adipogenic and/or myogenic pathways19,20,24. Recent studies revealed two subpopulations of pericytes, with different marker expression and dist...

Muscular dystrophy is a muscle-degenerative disorder that has no effective treatments so far. The development of therapies that promote tissue regeneration has always been of great interest. Tissue regeneration and repair after damage depend on resident stem cells/progenitor cells1. Satellite cells are committed myogenic precursor cells that contribute to muscle regeneration2,3,4,5,6,7. Their clinical use, however, is hampered by their limited migration and low survival rate after injection, as well as by their decreased differentiation capability after in vitro amplification8,9,10,11. In addition to satellite cells, skeletal muscles also contain many other cell populations with myogenic potential12,13,14,15,16, such as platelet-derived growth factor receptor-beta (PDGFR&#946;)-positive interstitial cells. There is evidence showing that muscle-derived PDGFR&#946;+ cells are able to differentiate into myogenic cells and improve muscle pathology and function14,17,18,19,20. PDGFR&#946; predominantly labels pericytes21, which are perivascular cells with pluripotency22,23. In addition to PDGFR&#946;, many other markers, including Neuron-Glial 2 (NG2) and CD146, are also used to identify pericytes21. It should be noted, however, that none of these markers is pericyte-specific21. Recent studies revealed two subtypes of muscle pericytes, called type I and type II, which express different molecular markers and carry out distinct functions19,24,25. Biochemically, type I pericytes are NG2+Nestin-, while type II pericytes are NG2+Nestin+19,24. Functionally, type I pericytes can undergo adipogenic differentiation, contributing to fat accumulation and/or fibrosis, whereas type II pericytes can differentiate along the myogenic pathway, contributing to muscle regeneration19,24,25. These results demonstrate that: (1) type I pericytes may be targeted in the treatment of fatty degenerative disorders/fibrosis, and (2) type II pericytes have great therapeutic potential for muscular dystrophy. Further investigation and characterization of these populations require an isolation protocol that enables the separation of type I and type II pericytes at a high level of purity.
Currently, the isolation of pericyte subpopulations relies on NG2-DsRed and Nestin-GFP double-transgenic mice19,24. The availability of NG2-DsRed mice and the quality of most NG2 antibodies limit the widespread use of this method. Given that all NG2+ pericytes also express PDGFR&#946; in skeletal muscles19,20,24, we hypothesize that NG2 can be replaced by PDGFR&#946; for the isolation of pericytes and their subpopulations. This work describes a FACS-based protocol that uses PDGFR&#946; staining and the Nestin-GFP signal. This method is less demanding for investigators because: (1) it does not require the NG2-DsRed background and (2) it uses commercially available PDGFR&#946; antibodies, which are well-characterized. In addition, it enables the simultaneous isolation of type I and type II pericytes at high purity, allowing for further investigation into the biology and therapeutic potential of these pericyte subpopulations. Following purification, these cells can be grown in culture, and their morphologies can be visualized. This work also shows that type I and type II pericytes isolated using this method, like those purified from double-transgenic mice, are adipogenic and myogenic, respectively.

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			<td height="22" style="height:22px;width:311px;">Cell Sorter</td>
			<td style="width:173px;">Sony</td>
			<td style="width:133px;">SH800</td>
			<td style="width:88px;"></td>
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			<td height="22" style="height:22px;width:311px;">Automatic Setup Beads</td>
			<td style="width:173px;">Sony</td>
			<td style="width:133px;">LE-B3001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">DMEM</td>
			<td style="width:173px;">Gibco</td>
			<td style="width:133px;">11995</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Avertin&#160;</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:133px;">T48402</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Pericyte Growth Medium</td>
			<td style="width:173px;">ScienCell</td>
			<td style="width:133px;">1201</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">MSC Basal Medium (Mouse)</td>
			<td style="width:173px;">Stemcell Technologies</td>
			<td style="width:133px;">5501</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Adipogenic Stimulatory Supplement (Mouse)</td>
			<td style="width:173px;">Stemcell Technologies</td>
			<td style="width:133px;">5503</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Fetal Bovine Serum</td>
			<td style="width:173px;">Gibco</td>
			<td style="width:133px;">16000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Horse Serum</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:133px;">H1270</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Collagenase Type 2</td>
			<td style="width:173px;">Worthington</td>
			<td style="width:133px;">LS004176</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">0.25% Trypsin/EDTA&#160;</td>
			<td style="width:173px;">Gibco</td>
			<td style="width:133px;">25200</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Penicillin/Streptomycin</td>
			<td style="width:173px;">Gibco</td>
			<td style="width:133px;">15140</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">PDL</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:133px;">P6407</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">PDGFR&#946;-PE Antibody</td>
			<td style="width:173px;">eBioscience</td>
			<td style="width:133px;">12-1402</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Perilipin Antibody</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:133px;">P1998</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">S-Myosin Antibody</td>
			<td style="width:173px;">DSHB</td>
			<td style="width:133px;">MF-20</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Alexa 555-anti-rabbit antibody&#160;</td>
			<td style="width:173px;">ThermoFisher Scientific</td>
			<td style="width:133px;">A-31572</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Alexa 555-anti-mouse antibody</td>
			<td style="width:173px;">ThermoFisher Scientific</td>
			<td style="width:133px;">A-31570</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Mounting Medium with DAPI</td>
			<td style="width:173px;">Vector Laboratories</td>
			<td style="width:133px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">DAPI</td>
			<td style="width:173px;">ThermoFisher Scientific</td>
			<td style="width:133px;">D1306</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">HEPES</td>
			<td style="width:173px;">Gibco</td>
			<td style="width:133px;">15630</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">EDTA</td>
			<td style="width:173px;">Fisher</td>
			<td style="width:133px;">BP120</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">BSA</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:133px;">A2058</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:311px;">NH4Cl</td>
			<td style="width:173px;">Fisher Scientific</td>
			<td style="width:133px;">A661</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:311px;">KHCO3</td>
			<td style="width:173px;">Fisher Scientific</td>
			<td style="width:133px;">P184</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">PBS</td>
			<td style="width:173px;">Gibco</td>
			<td style="width:133px;">14190</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">18G Needles</td>
			<td style="width:173px;">BD</td>
			<td style="width:133px;">305196</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">10ml Serological Pipette</td>
			<td style="width:173px;">BD</td>
			<td style="width:133px;">357551</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of type i and type ii pericytes from mouse skeletal muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

FACS parameters, including laser intensity and channel compensation, are corrected based on the results of unstained control and single-color controls. The PDGFR&#946;-PE-FMO control is used to set the gating for the PDGFR&#946;-PE+ population (Figure 1A). Among the PDGFR&#946;-PE- cells, two populations representing Nestin-GFP+ and Nestin-GFP- cells are clearly separated (Figure 1A). G...

Watch this Scientific Journal Video about Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles at JoVE.com

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

The overall goal of this procedure is to isolate sub-populations of pericytes from skeletal muscles using FACS. This method can help answer chief questions in the field of pericyte biology, such as what is the biological difference between type I and type II pericytes? The main advantage of this protocol is that it allows simultaneous isolation of type I and type II pericytes without use of NG2 dsRED transgene.The applications of this protocol are not limited to skeletal muscles. It may also be used to isolate pericytes from other organs, such as the brain. To begin, first obtain the desired tissue from a nestin-GFP transgenic mouse, and then wash the tissue twice in ice-cold PBS containing antibiotics.Then transfer the samples to a sterile 10-centimeter plate. Next, using sterile scissors and blades, cut the muscles into fine pieces of one to two millimeters cubed. If needed, add a small amount of DMEM to prevent the tissue from drying out.Pipette the tissue up and down through a 10-millimeter serological pipette to disrupt it mechanically. Then, add fresh digestion solution, DMEM supplemented with 0.2%Type 2 collagenase, to the mixture, and incubate the sample at 37 degrees Celsius for two hours at 35 RPM. Following two hours of incubation, titrate the mixture with an 18-gauge needle and centrifuge the resulting suspension at 500 x g for five minutes.Then, re-suspend the pellet in 0.25%trypsin-EDTA and incubate the sample at 37 degrees Celsius for 10 minutes. Next, add 10 milliliters of DMEM supplemented with 20%FBS, and centrifuge the sample at 500 x g for five minutes. Once pelleted, re-suspend the cells in 10 milliliters of red blood cell lysis buffer and spin the suspension at 500 x g for five minutes.Discard the supernatant and re-suspend the pellet in 10 milliliters of sorting buffer. Then, filter the mixture through a 40-micrometer cell strainer, and centrifuge the obtained single-cell suspension at 500 x g for five minutes once more. Re-suspend the pelleted cells in one milliliter of sorting buffer, count them with the hemocytometer, and further dilute to obtain the suspension of five times 10 to the sixth cells per milliliter.Prior to cell sorting, prepare controls and stain the samples as described in the text protocol. Turn on the sorter and the software, and when asked, scan and insert a 100-micron sorting chip. Then, load automatic setup beads to perform an automatic setup.Once the automatic setup is completed, open the Experiment tab, and click on Blank Template. Next, in Measurement Settings, enter DAPI for FL1, Nestin-GFP for FL2, and PDGFR-beta for FL3, and uncheck the boxes for the remaining channels. Activate 405, 488, and 561 lasers, and click on Create New Experiment.Then, click on the Start Compensation Wizard option, and follow the Compensation Wizard software commands. After loading the unstained control, click on Start, and select Detector Threshold Settings to set the sensor gain of the FSC and BSC detectors, and to visualize the population on the scale. Adjust the gain levels of the fluorescence channels to position the negative population on the left side of the histogram, and record data by clicking the Record button.When asked by the software, load the single-color control, and record data by clicking Start and Record. Then, gate positive population in the histogram and click Next. To perform compensation, open Compensation tab, select Calculate Matrix, then open Calculate Compensation Settings panel and click Calculate.Once the calculation is completed, click Finish to close the Compensation Wizard. Now that the compensation has been performed, load the FMO control for PDGFR-beta staining. Click Start.In the All Events plot, double-click on Gate A to generate a daughter population plot. Then, assign y-axis of the daughter plot to DAPI, and draw Gate B comprising only viable cells. After creating another daughter plot, assign its x-axis to FSC-H, and y-axis to FSC-W, and draw Gate C around the population of single cells.For Gate C, generate a daughter plot of PDGFR-beta versus Nestin-GFP. Then, click the Record button to record data. Finally, load the sample, record the data, and then click on Pause to preserve the sample.Set the gating boundaries for PDGFR-beta positive and Nestin-GFP positive cells, and create gates for Type I and Type II pericytes populations. Here shows the gating boundaries for Type I and Type II pericytes in PDGFR-beta-PE-FMO Control. In the Sorting Method tab, select Two Way Tubes and assign type I and type II pericytes to the left and right collecting tubes.Place 15-milliliter collecting tubes pre-filled with five milliliters of sorting buffer on the collection stage, and click the Load Collection button. Click the Resume button to run the sample, and Start Sort to collect the cells. Once sorted, centrifuge the cells at 500 x g for five minutes.Re-suspend the resulting pellet in one milliliter of pericyte medium, and count the cells in a hemocytometer. Seed pericytes of both types in a 24-well plate containing coverslips pre-coated with poly-D-lysine at a density of approximately one times 10 to the fourth cells per centimeter squared. Culture the cells in 0.5 milliliters of pericyte medium for three days at 37 degrees Celsius and 5%carbon dioxide.After 3 days of incubation, examine pericyte morphology, and, under a fluorescent microscope, assess their endogenous expression of nestin GFP. Then, replace the pericyte medium with 0.5 milliliters of adipogenic or myogenic medium to initiate cell differentiation. Change the medium every two to three days.After 14 days of differentiation, stain the pericytes to visualize perilipin and s-myosin and image the cells under the fluorescent microscope. Presented here are microscopic images of type I and type II pericytes that, following the isolation, were grown in pericyte medium for three days. Type I pericytes were characterized by the round cell bodies with short projections and lack of the endogenous GFP expression.Type II pericytes had small cell bodies with long and thin processes, and had shown endogenous GFP expression. When both cell types were cultured in a dipogenic medium, type I pericytes differentiated into adipocytes, as evidenced by the pronounced expression of an adipocyte marker, perilipin. On the contrary, Type II pericytes did not respond to a dipogenic differentiation.Finally, pericytes of both types revealed distinct responses to myogenic medium. Type I pericytes did not undergo differentiation into myocytes, while type II pericytes formed myotubes, as evidenced by s-myosin-positive staining, confirming their myogenic activity. While attempting this procedure, it's important to include FMO controls to determine the gating boundaries.After watching this video, you should have a good understanding of how to isolate type I and type II pericytes from skeletal muscle by fluorescence-activated cell sorting.

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9.1K Görüntüleme.  University of Minnesota. The overall goal of this procedure is to isolate sub-populations of pericytes from skeletal muscles using FACS. This method can help answer chief questions in the field of pericyte biology, such as what is the biological difference between type I and type II pericytes? The main advantage of this protocol is that it allows simultaneous isolation of type I and type II pericytes without use of NG2 dsRED transgene.The applications of this protocol are not limited to skeletal muscles. It ma...