Anti cd31 apc

Fc regions on cells were blocked with 2 µl Fc-block (BD Pharmingen, 553141) in 50 µl FACS buffer per 10⁶ cells for 10 min on ice. For fibroblast negative selection, the cells were incubated in staining cocktail containing anti-CD31-APC (1 µl/10⁶ cells, BD Biosciences, 561814, Clone: MEC 13.3), anti-CD45-APC (1 µl/10⁶ cells, BD Biosciences, 559864, Clone: 30-F11), anti-CSPG4-AF647 (0.4 µl/10⁶ cells, Bioss, bs-4800R-A647), and anti-CD326-APC (5 µl/10⁶ cells, BD Biosciences, 563478, Clone G8.8) in FACS buffer for 30 min on ice. 4′-6′-diamidino-2-phenylindole (DAPI) was added to the cell suspension before sorting. We gated on DAPI⁻, Epcam⁻, CD31⁻, CD45⁻, NG2⁻ cells after excluding doublets. The cells were sorted using a FACSARIA II (BD Biosciences) into individual wells of 386-well plates containing lysis buffer provided by the Eukaryotic single cell facility (ESCG), SciLifeLabs, (Stockholm, Sweden). For population resorting, cells obtained from MMTV-PyMT mice crossed with the PdgfRα-EGFP reporter mouse^{42 (link)} were stained with anti-CD31-APC, anti-CD45-APC, anti-NG2-AF647, anti-CD326-APC, anti-PDGF-Rβ-biotin (Thermo Scientific, 13-1402-82) for 30 min on ice, followed by 20 min incubation on ice with streptavidin-PE/Cy7 (Thermo Scientific, 25-4317-82).

Both Aged and Young MSCs were characterized by their cell surface antigen expression and trilineage differentiation capability using flow cytometry. Antibodies used for MSC characterization were anti-CD105-APC (17-1057-41; Thermo Fisher Scientific), anti-CD44-APC (17-0441-81; Thermo Fisher Scientific), anti-CD73-APC (17-0739-41; Thermo Fisher Scientific), anti-CD90-APC (17-0909-41; Thermo Fisher Scientific), anti-CD45-APC (17-9459-41; Thermo Fisher Scientific), and anti-CD31-APC (17-0319-41; Thermo Fisher Scientific).

Using 0.2% II type collagenase (Sigma, USA), inflamed TA muscles were collected and digested for 40 min at the condition of 37 °C. In vivo, the single cell suspension obtained from muscle homogenate was blocked. In vitro, cultured cells were digested with Trypsin (Sigma, USA), resuspended in ice cold PBS to obtain the single cell suspension. The following fluorescent antibodies were used: anti-CD45-Pacific Blue, anti-F4/80-PE, anti-CD11b-PE, anti-MHC-II-eFluor 450, anti-Ly6C-FITC, anti-CX3CR1-APC, anti-CD206-eFluor 700, anti-Bcl3-FITC, anti-CD31-APC, anti-IL-10-FITC, rabbit anti-p-STAT3-FITC, the antibodies above were purchased from ThermoFisher and their dilution ratios were 1:100; Other antibodies involved anti-Vav1-FITC (1:100, Biorbyt, USA), anti-Rac1-GTP-FITC (1:100, Proteintech, USA), anti-Tunel-FITC (5:50, Yeason, China), anti-Annexin-V-APC (5:100, Sigma, USA), anti-CRT-Alexa Fluor 647 (1:100, Abcam, UK), anti-PKH67-Alexa Fluor 647 (1:250, Zeye, China), anti-CD36-Alexa Fluor 700 (1:100, eBioscience, USA), and anti-PPARγ-FITC (1:100, Abcam, UK). To analyze the labeled cells, FACSAria II cell sorter with FlowJo software (BD Biosciences, USA) were used.