Satellite cells were isolated as previously described [27 (link)] from hindlimb muscles. Cells were labeled using the following antibodies: 1:400 CD31-PE (clone 390; eBiosciences), 1:400 CD45-PE (clone 30-F11; BD Pharmingen), 1:4000 Sca-1-PE-Cy7 (clone D7; BD Pharmingen), 1:500 α7-integrin-APC (Clone R2F2; AbLab). Dead cells were excluded by propidium iodide (PI) staining. Cell sorting was performed using a BD FACSAria II cell sorter (Becton-Dickinson). Sorted cells were immediately reanalyzed to ensure purity. Analyses of flow cytometry data were performed using FACSDiva (BD version 8.0.1).
Cd31 pe clone 390
Manufactured by Thermo Fisher Scientific
CD31-PE (clone 390) is a fluorescently labeled antibody that binds to the CD31 (also known as PECAM-1) protein. CD31 is expressed on the surface of endothelial cells, platelets, and certain leukocyte populations. This product can be used to identify and analyze cell populations expressing CD31 in flow cytometry applications.
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Lab products found in correlation
Sca 1 pe cy7 clone d7, by BD (3 mentions)
Cd45 pe clone 30 f11, by BD (3 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Brdu flow kit, by BD (1 mentions)
Facsymphony a3 flow cytometer, by BD (1 mentions)
Facs lsr 2, by BD (1 mentions)
Bromodeoxyuridine (brdu), by BD (1 mentions)
Donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Cd45 fitc clone 30 f11, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Collagenase d, by Roche (1 mentions)
4 protocols using cd31 pe clone 390
1
Isolation and Characterization of Satellite Cells
2
Assessing Stem Cell Proliferation in Muscles
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To compare the proliferative abilities of SCs in pharyngeal and hindlimb muscles in vivo, Bromo-2′-deoxyuridine (BrdU) assays were performed. Three-month-old male mice were injected with 10 µg BrdU (Sigma-Aldrich, St. Louis, MO)/g body weight intraperitoneally every 12 h for 2 days before sacrifice. Muscles were dissected and digested as described above. To assess proliferation, isolated mononucleated cells from pharyngeal or gastrocnemius muscles were immunostained with the following antibodies: 1:400 CD31-PE (clone 390; eBiosciences, San Diego, CA), 1:400 CD45-PE (clone 30-F11; BD Biosciences, San Jose, CA), 1:4000 Sca-1-PE-Cy7 (clone D7; BD Biosciences, Vancouver, Canada), and 1:20 a7-integrin-APC (FAB3518A; R&D SYSTEMS, Minneapolis, MN). Subsequently, cells were labeled for BrdU using a BrdU flow kit (BD biosciences, Vancouver, Canada), and proliferating SCs and FAPs were collected according to the following sorting criteria, CD31-/CD45-/Sca1-/Intergrin7α+/BrdU+ and CD31-/CD45-/Sca1+/BrdU+, respectively using BD FACS LSR II or BD FACSymphony A3 flow cytometer (flow rate: 300–1,000 event/sec). Single cells were selected by FSC-A vs. FSC-H and then selected by SSC-A vs. SSC-H (Supplementary Figure S2A ). Gating for SCs, FAPs, BrdU was drawn by FMO samples (Supplementary Figures S2B,C ). Cells from gastrocnemius muscles were used as limb muscle controls.
3
Quantifying Muscle Stem Cells and Progenitors
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To analyze the number of satellite cells and fibroadipose progenitor cells (FAPs) in injured muscles, muscles were dissected and digested with dispase II and collagenase II as previously described.[53 (link)
] Isolated mononucleated cells were immunostained with the following antibodies: 1:400 CD45‐PE (clone 30‐F11; BD Biosciences), 1:4000 Sca‐1‐PE‐Cy7 (clone D7, BD Biosciences), 1:400 CD31‐PE (clone 390; eBiosciences). Fibroadipose progenitor cells were counted using the following criteria: CD31−/CD45−/Sca1+ and satellite cells are counted by tdTomato+ using BD LSR II cytometry analyzer and analyzed using FCS Expression 6 Flow software 6.01.
] Isolated mononucleated cells were immunostained with the following antibodies: 1:400 CD45‐PE (clone 30‐F11; BD Biosciences), 1:4000 Sca‐1‐PE‐Cy7 (clone D7, BD Biosciences), 1:400 CD31‐PE (clone 390; eBiosciences). Fibroadipose progenitor cells were counted using the following criteria: CD31−/CD45−/Sca1+ and satellite cells are counted by tdTomato+ using BD LSR II cytometry analyzer and analyzed using FCS Expression 6 Flow software 6.01.
4
Isolation and Characterization of Lung Cells
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Lungs were harvested, cut into small pieces, and digested (1 hour, 37°C) with 4 mg/mL collagenase D (Roche). Tissue was meshed through a 40 μm cell strainer, and RBCs were lysed. Cells were stained using the following antibodies (eBioscience, Thermo Fisher Scientific): CD45-FITC (clone 30-F11), CD31-PE (clone 390), and CD326-PerCP (clone G8.8). Cells were defined as endothelial cells (CD45–CD31+) and epithelial cells (CD45–CD31–CD362+). For ACE2 receptor staining, cells were stained using goat anti-mACE2 antibody (R&D Systems, Bio-Techne, AF3437) followed by donkey anti-goat IgG coupled to AF647 (Thermo Fisher Scientific, A20186). For TMPRSS2 staining, cells were stained using anti-TMPRSS2 polyclonal antibody coupled to AF647 (Santa Cruz Biotechnology, sc-515727). Cells were analyzed on FACSCalibur (BD Biosciences).
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