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).
Cd45 pe clone 30 f11
Manufactured by BD
CD45-PE (clone 30-F11) is a fluorescently-labeled antibody that binds to the CD45 cell surface antigen. CD45 is a protein tyrosine phosphatase expressed on the surface of most hematopoietic cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd31 pe clone 390, by Thermo Fisher Scientific (3 mentions)
Sca 1 pe cy7 clone d7, by BD (3 mentions)
Axio observer z1, by Zeiss (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)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Clone ck3 6h5, by Miltenyi Biotec (1 mentions)
Clone cam5, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Triton x 100, by Merck Group (1 mentions)
4 protocols using cd45 pe clone 30 f11
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
Multiparametric Profiling of Cell Populations
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After Vortex processing, cells were collected in a cell culture-treated 96-well plate or a PolySorp-coated eight-well strip (Nunc), fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 10 min, permeabilized with 0.4% v/v Triton X-100 (Sigma Aldrich) for 7 min, blocked with 10% goat serum (Invitrogen) for 30 min, and labeled for 1 h at room temperature (RT) with 4,6-diamidino-2-phenylindole (DAPI; Life Technologies), anti-CD45-phycoerythrin antibody (CD45-PE, clone HI30, BD Biosciences), and a cocktail of primary antibodies labeled with fluorescein isothiocyanate to identify cytokeratin (CK)-positive cells (clone CK3-6H5, Miltenyi Biotec, and clone CAM5.2, BD Biosciences). 28 For experiments with healthy mouse blood, a similar staining protocol was optimized but with a rat anti-mouse CD45-PE antibody (CD45-PE, clone 30-F11, BD Pharmingen). For patient-derived orthotopic xenograft (PDOX) samples, an anti-CK-AlexaFluor (AF) 488 antibody (clone AE1/AE3, eBioscience) and an antivimentin-AF647 antibody (clone V9, Abcam) were used in addition to the previously listed antibodies. After staining, the cells were imaged (Axio Observer Z1, Zeiss) and manually enumerated using specific classification criteria previously published. 27 Capture performance was evaluated as follows:
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