Multiparametric Flow Cytometry for Endothelial Cell Analysis

Cells were labeled with CD31-FITC antibody (0.5 μg/sample) to validate EC isolation (BD-Pharmigen, 558738) and incubated on ice for 30 min. Cells were fixed in ice-cold 1% PFA for 10 min. For EdU cell cycle phase determination, we used the Click-iT EdU Pacific Blue flow cytometry assay kit, per manufacturer recommendation (ThermoFisher, C10418). Cells were analyzed on a BD-FACSymphony Flow Cytometer. For iH2B-FT cell flow analysis, only live cells were processed for cell cycle speed determination to avoid photoconversion ^{45 (link)}. For imaging flow cytometry (IFC), cells were labeled with CD31-FITC and fixed, as described above. Cells were then permeabilized with saponin buffer (0.1% saponin, 0.5% BSA in PBS) and stained for intracellular p-RB1 [Cell Signaling Technology, # 8156; 1:500 in flow buffer (0.5% BSA in PBS)] for 1h. Cells were centrifuged at 1200×g at 4°C and washed twice in ice-cold PBS. Cells were stained with Alexa Fluor 532 secondary antibody (ThermoFisher, A20182; 1:1000 in flow buffer) and centrifuged at 1200×g at 4°C and washed three times in ice-cold PBS before a final resuspension of approximately 100,000 cells/tube in DAPI-supplemented PBS for final analysis. Cells were analyzed on an Amnis ImageStream cytometer. Analyses were performed using IDEAS software and the bright detail intensity feature was used.