Cd31 antibody

The single cell suspensions were filtered with screen cloth and cell surface staining was performed in FACS buffer containing CD31 antibody (Cat# 303102, BioLegend, USA) on ice for 1 h. Following washing twice with FACS buffer, the percentages of EC subtype (CD31 positive) in these single cell suspensions were detected using a FACS flow cytometry system (Cytoflex, Beckman Coulter, USA).

For flow cytometry characterization, the cell pellet was resuspended in 100 μL of sorting buffer with the anti-feeder cells antibody (mEFSK4 clone) (Miltenyi, 1:100) and CD31 antibody (Biolegend, 1:100). Unlabeled cells were used as a negative control for antibody labeling. After a 15 min incubation at R/T in the dark, samples were washed twice with FACS buffer and spun at 650 g for 5 min. Then, the supernatant was discarded, and the final pellet was resuspended in 250 μL of FACS buffer. Just before flow cytometry, 7-aminoactinomicina D (7-AAD) (Thermo Scientific) was added to each sample to determine viability. Standard, strict forward scatter width versus area criteria were used to discriminate doublets and gate only singleton cells. Viable cells were identified by staining with 7-AAD. Viable cells gated on the FSC/SCC were analyzed on the basis of the expression of mEFSK4 and CD31 antibodies as well as tdTomato/ZsGreen reporter protein expression in each subpopulation. Flow cytometry for these samples was performed on a CytoFLEXLX Flow Cytometer (Beckman Coulter, Life Sciences). The files generated by flow cytometry were processed using FlowJo Software (Tree Star, Ashland, USA).

Podocyte isolation was performed as described previously [39 (link)]. After red blood cell removal using ammonium chloride potassium lysis buffer and endothelial cell depletion using CD31 antibody (Biolegend, San Diego, CA, USA), nephrin-positive cells were prepared from minced murine kidneys using magnet-activated cell sorting with nephrin antibody (Thermo Fisher Scientific, Waltham, MA, USA).