Anti sirpa pe cy7 clone se5a5

For cell-surface antigens, staining was carried out in PBS with 3% FCS. For intracellular antigens, staining was carried out on cells fixed with 4% paraformaldehyde in PBS. Staining was done in PBS with 3% FCS and 0.5% saponin (Sigma). Cells were stained at a concentration of 2.5 × 10⁶ cells/ml with anti-KDR- APC (R&D Systems; 1:10) and anti-PDGFRA– PE (R&D Systems; 1:20), anti-SIRPA–PE-Cy7 (clone SE5A5; BioLegend; 1:500), anti-Podoplanin-PE (BioLegend, 1:200), anti-CD90-APC (BD Pharmingen, 1:2000), anti-PDGFRβ-PE (BD Pharmingen, 1:200), anti-EPCAM-PE (BD Pharmingen, 1:200), anti-CD31-PE (BD Pharmingen, 1:10), anti-CTNT (clone 13-11; Thermo NeoMarkers; 1:400), goat anti-mouse IgG–APC(BD; 1:200). Cells assayed for Aldefluor (STEMCELL Technologies) were prepared based on manufacturers instructions. Incubation with the Aldefluor reagent was 45 minutes. Stained cells were analyzed on an LSRII flow cytometer (BD Biosciences). For FACS, the cells were sorted at a concentration of 10⁶ cells/ml in IMDM/5% FCS using a FACSAriaTMII (BD Biosciences) cell sorter (SickKids-UHN Flow Cytometry Facility). Data were analyzed using FlowJo software (Treestar).