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 skin with 70% ethanol. 
	NOTE: Tribromoethanol was used here instead of ketamine for anesthesia/euthanasia, as ketamine is known to interact with the NMDA receptors, which could potentially have an impact on the study.</li>
	<li>Place the mice in the supine position and use a scalpel to make a horizontal incision on the abdominal skin. Peel off the skin by hand, pulling in opposite directions to expose the hindlimb muscles.</li>
	<li>Collect the muscles from both hindlimbs using forceps and scissors. Store them in sterile phosphate-buffered saline (PBS) supplemented with 1% penicillin-streptomycin (P/S) on ice.</li>
	<li>Wash the dissected hindlimb muscles in ice-cold PBS supplemented with 1% P/S two times and transfer them to a sterile 10 cm plate.</li>
	<li>Carefully dissect out nerves, blood vessels, and connective tissue from the muscles using forceps and scissors under a dissection microscope at 2X magnification.</li>
	<li>Finely chop and mince the muscles into small pieces (1-2 mm3) using sterile scissors and blades. If necessary, add a small amount of Dulbecco&#39;s Modified Eagle Medium (DMEM) to ensure that the muscles are not dried out.</li>
	<li>Mechanically break down the tissue by pipetting up and down through a 10 mL serological pipette 10 times.</li>
	<li>Add freshly made digestion solution (DMEM supplemented with 0.2% Type 2 collagenase) to the mixture. Incubate at 37 &#176;C for 2 h with gentle agitation at 35 revolutions/min.</li>
	<li>Triturate using an 18 G needle to homogenize the mixture. Then, centrifuge at 500 x g for 5 min. Discard the supernatant and then resuspend the pellet in 0.25% trypsin/EDTA. Incubate at 37 &#176;C for 10 min. Repeat the centrifugation step two more times.</li>
	<li>Add 10 mL of DMEM supplemented with 20% fetal bovine serum (FBS) to the solution and centrifuge at 500 x g for 5 min. Discard the supernatant and resuspend the pellet in red blood cell lysis buffer (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA).</li>
	<li>Centrifuge at 500 x g for 5 min. Discard the supernatant and resuspend the pellet in sorting buffer (20 mM HEPES, pH 7.0; 1 mM EDTA; and 1% BSA in Ca/Mg2+-free PBS, pH 7.0). Filter the mixture through a 40 &#181;m cell strainer to obtain a single-cell suspension.</li>
	<li>Centrifuge at 500 x g for 5 min. Discard the supernatant and resuspend the pellet in 1 mL of sorting buffer.</li>
	<li>Count the cell number with a hemocytometer and dilute the single-cell suspension to 5 x 106/mL in sorting buffer.</li>
</ol>
2. Cell Staining and Sorting
<ol>
	<li>Prepare controls and the sample as described in Table 1. Stain the single-cell suspension with the respective antibodies on ice for 30 min, as described in Table 1.</li>
	<li>Centrifuge at 500 x g for 5 min and wash the pellets twice with sorting buffer.</li>
	<li>Add DAPI to the single-cell solutions, as indicated in Table 1. Use 5 &#181;g/mL DAPI (final concentration) for the DAPI single-color control and 1 &#181;g/mL DAPI for the PDGFR&#946;-PE-FMO control and the sample. Keep all tubes on ice throughout the experiment.</li>
	<li>Turn on the sorter and the software. Scan and insert a 100 &#181;m sorting chip when prompted.</li>
	<li>Perform the automatic setup (i.e., chip alignment, droplet calibration, side stream calibration, and sort delay calibration) by loading the automatic setup beads when prompted.</li>
	<li>When automatic setup is complete, go to the &#34;Experiment&#34; tab, click on &#34;New,&#34; and select the &#34;Blank Template&#34; from &#34;Public Templates.&#34;</li>
	<li>Under &#8220;Measurement Settings,&#8221; enter &#8220;DAPI&#8221; for &#8220;FL1,&#8221; &#8220;Nestin-GFP&#8221; for &#8220;FL2,&#8221; and &#8220;PDGFR&#946;&#8221; for &#8220;FL3.&#8221; Uncheck the boxes for &#8220;FL4&#8221;-&#8220;FL6&#8221;.</li>
	<li>Check the boxes to activate the 405, 488, and 561 lasers and click on &#34;Create New Experiment.&#34;</li>
	<li>Select the &#34;Start Compensation Wizard&#34; option and follow the &#34;Compensation Wizard&#34; software prompts to set up the compensation.
	<ol>
		<li>Load the unstained control and click &#34;Start.&#34; Click &#34;Detector &#38; Threshold Settings&#34; and adjust the sensor gain of the FSC and BSC detectors to place the population on the scale.</li>
		<li>Adjust the gain levels of the FL1-FL3 fluorescence channels to place the negative populations on the left side of the histograms. Click the &#34;Record&#34; button to record the data.</li>
		<li>Load single-color controls one by one when prompted. Click &#34;Start and Record&#34; to record the data. Adjust the gates for the positive populations on the histograms. Click &#34;Next.&#34;</li>
		<li>Go to &#34;Calculate Matrix&#34; on the &#34;Compensation&#34; tab and click &#34;Calculate&#34; in the &#34;Calculate Compensation Settings&#34; panel to perform the compensation. Click &#34;Finish&#34; to exit the &#34;Compensation Wizard.&#34;</li>
	</ol>
	</li>
	<li>Load the PDGFR&#946;-PE-FMO control and click &#34;Start.&#34; Draw a polygon gate (Gate A) around the cells of interest under the &#34;All Events&#34; plot.</li>
	<li>Double-click inside Gate A to create a child plot. Change the Y-axis to DAPI and draw a polygon gate (Gate B) around live (DAPIlow) cells. Double-click inside Gate B to create a child plot. Change the X-axis to FSC-H and the Y-axis to FSC-W and draw a polygon gate (Gate C) around the singlets to eliminate doublets.</li>
	<li>Double-click inside Gate C to create a child plot. Change the X-axis to Nestin-GFP and the Y-axis to PDGFR&#946;-PE. Click the &#8220;Record&#8221; button to record the data.</li>
	<li>Load the sample and repeat steps 2.11-2.12. After recording, click on &#34;Pause&#34; to preserve the sample.</li>
	<li>Define the gating boundaries for PDGFR&#946;+ and Nestin-GFP+ cells based on the PDGFR&#946;-PE-FMO control. Draw gates for the PDGFR&#946;+Nestin-GFP- and PDGFR&#946;+Nestin-GFP+ populations.</li>
	<li>Under &#8220;Sorting Method,&#8221; select &#8220;2-way Tubes&#8221; and assign &#8220;PDGFR&#946;+Nestin-GFP-&#8221; and &#8220;PDGFR&#946;+Nestin-GFP+&#8221; cells to the left and right collecting tubes, respectively. Mount the sorting buffer-filled 15 mL collecting tubes on the collection stage and click the &#8220;Load Collection&#8221; button&#34;.</li>
	<li>Click the &#34;Resume&#34; button to keep the sample running. Click &#34;Start Sort&#34; to collect PDGFR&#946;+Nestin-GFP- (type I pericytes) and PDGFR&#946;+Nestin-GFP+ (type II pericytes) cells.</li>
</ol>
3. Post-sorting Analyses
<ol>
	<li>Centrifuge the sorted cells at 500 x g for 5 min, resuspend the pellet in 1 mL of pericyte medium (see Materials Table), and count the cell density using a hemocytometer.</li>
	<li>Seed type I and type II pericytes on poly-D-lysine (PDL)-coated coverslips at ~1 x 104 cells/cm2. Grow in pericyte medium for 3 days at 37 &#176;C with 5% CO2.</li>
	<li>On day 3, examine the pericyte morphology (under phase contrast) and endogenous Nestin-GFP expression using a fluorescent microscope (excitation laser: 488 nm, excitation filter: 470/40 nm, and emission filter: 515/30 nm). Take images under a 20X objective (0.45 NA).</li>
	<li>Replace the pericyte medium with adipogenic (mouse MSC basal medium + adipogenic stimulatory supplement) and myogenic (DMEM + 2% horse serum) medium to initiate adipogenic and myogenic differentiation, respectively, as described previously20. Change the medium every 2-3 days.</li>
	<li>Fix the cells on day 17 (14 days after adipogenic/myogenic differentiation) in 4% paraformaldehyde (PFA) for 20 min at room temperature. 
	NOTE: Caution, PFA is a carcinogen.</li>
	<li>Perform immunocytochemistry against perilipin (adipocyte marker) and S-myosin (mature myotube/myofiber marker), as described in previous publications19,20.
	<ol>
		<li>Wash the fixed cells 3 times in PBS for 10 min at room temperature.</li>
		<li>Add blocking buffer (PBS supplemented with 5% donkey serum, 3% BSA, and 0.3% Triton X-100) and incubate at room temperature for 1 h.</li>
		<li>Incubate the cells with anti-perilipin (2 &#181;g/mL) and/or anti-S-myosin (2 &#181;g/mL) antibodies at 4 &#176;C overnight.</li>
		<li>Wash the cells 3 times in PBS for 10 min at room temperature.</li>
		<li>Incubate the cells with Alexa 555 donkey anti-rabbit (4 &#181;g/mL) and/or Alexa555 donkey anti-mouse (4 &#181;g/mL) antibodies at room temperature for 1 h.</li>
		<li>Wash the cells 3 times in PBS for 10 min at room temperature.</li>
		<li>Mount the immunostained cells with mounting medium containing DAPI (see the table of materials). Examine perilipin and S-myosin expression using a fluorescent microscope (excitation laser: 543 nm; 540/45 nm excitation and 600/50 nm emission) and take images under a 40X objective (0.60 NA).</li>
	</ol>
	</li>
</ol>

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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. 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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=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. 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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.

<table border="0" cellpadding="0" cellspacing="0" width="705">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<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>
		</tr>
		<tr height="22">
			<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 ...

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9.1K Views.  University of Minnesota. This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

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

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...