Anti cd45 bv711

Skeletal muscle tissue dissociation by enzymatic digestion and isolation of FAPs by FACS with anti-CD140a (PDGFRA)–APC (eBioscience, 17-1401-81) and anti-Ly6A/E (SCA-1)-v450 (BD Biosciences, 560653) was conducted as previously described (6 (link), 12 (link), 18 (link), 35 (link)), with the following modification: magnetic depletion of CD31⁺ and CD45⁺ cells prior to FACS was omitted, and CD31⁺ and CD45⁺ cells were detected with anti-CD31-bv711 (BD Biosciences, 740690; 1:800) and anti-CD45-bv711 (BD Biosciences, 563709; 1:500) antibodies. Sorting was performed on a FACSAria II (BD Biosciences) equipped with 405, 488, 561, and 633 nm lasers.
FACS-isolated FAPs were grown on tissue culture flasks (Nunc) in DMEM (Thermo Fisher Scientific, 11995065) with 50 U/mL penicillin and 50 μg/mL streptomycin (Pen/Strep; Gibco) and 20% premium FBS (Atlanta Biologicals, lot C19032) as previously described (6 (link), 12 (link)). FAPs were maintained at 37°C in a humidified atmosphere at 5% CO₂. Media were changed every other day. All experiments utilized FAPs that had undergone less than 7 population doublings (approximately 3 passages).

RTMs were collected at 5 × 10⁵ per FACS tube, washed with FACS buffer (1% BSA/5 mM EDTA/PBS, pH 7.2), and incubated in blocking buffer (1:100 anti-CD16/CD32 in FACS buffer) for 15 min at 4°C. After blocking, RTMs were washed with FACS buffer and stained by anti-CD45-BV711 (BD), F4/80-PE (BioLegend), CD11b-FITC (BD), Siglec-F-PE (BD), CD11c-APC-Cy7 (BioLegend), or CD80-APC (BD) at a dilution of 1:200 by FACS buffer for 30 min at 4°C. After washing with FACS buffer, cells were analysed by LSRFortessa X-20 (BD) and FlowJo software, v 10.8.1.