Anti pdgfrα apc

Stromovascular cell (SVC) fractions from mouse gWAT were isolated, as previously described [6] (link). For EdU detection, fixed SVCs were processed for Click-it reaction first, followed by cell-surface marker staining. Antibodies used for flow cytometry analysis were the following: anti-PDGFRα-APC (Biolegend, cat # 135907) for ASCs, CD44-FITC (Biolegend, cat # 103021), and F4/80-APC (Biolegend, cat # 123115) for ATMs. Analytic cytometry was performed using BD LSR III (BD Biosciences) flow cytometers. Raw data were processed using FlowJo software (Tree Star). For gene expression analyses by qPCR or RNAseq, ATMs and ASCs were isolated by magnetic cell sorting (MACS) with anti-F4/80-FITC/anti-FITC-microbeads and anti-PDGFRα-PE/anti-PE-microbeads, respectively (Miltenyi Biotech).

D4 EBs were disassociated with Accutase (Biolegend) and washed with PBS. Then 1 × 10⁶ cells were incubated with anti–FLK-1-PE (Biolegend, 121905) and anti–PDGFR-α-APC (Biolegend, 135907) at 4 °C for 30 minutes and washed with PBS. Cells were sorted using a Beckman CytoFlex Sand analyzed with FlowJo software (v.7.6).