Fluorescence-activated cell sorting (FACS)

FACS was performed to isolate ECs from the heart and kidney. Both organs were digested using tissue-specific protocols of GentleMACS Dissociator in Hank’s balanced salt solution (HBSS) (H9269, Sigma-Aldrich®, Portugal) containing collagenase II (CLS-2, Worthington, USA) at 6, 5 mg/ml concentration for heart and collagenase IV (CLS-4, Worthington, USA) at 1mg/ml concentration for kidney. Both solutions had DNase (A3778, VWR, Portugal) at 60U/ml.

The suspension was filtered and the cellular portion was collected. The cellular suspension was mixed with HBSS with 10% FBS to neutralize enzymatic activity and centrifuged at 300xg for 10 minutes. Erythrocyte depletion was accomplished by osmotic shock as deyonized water was added to the pellet and kept under stirring for 11 seconds. FACS medium (0.01% sodium azide and 3% FBS in PBS) was immediately added to interrupt osmolysis and cells were centrifuged again. Cells were evenly distributed for each staining in a round bottom multi-well plate and incubated during 30 min with the respective antibody mix: CD31-PeCy7 (Rat IgG2a; eBiosciences - 25–0311), CD31-APC (Rat IgG2a; Biolegend – 102516); CD45-PeCy7 (Rat igG2b, k; eBiosciences - 25–0451), CD45-PE (Rat IgG2b, k; eBioscience - 12–0451-82); CD54-APC (Rat IgG2b, κ; Biolegend – 116120), CD90-FITC (Rat IgG2b, k; Biolegend – 105305);, TER119-PeCy7 (Rat IgG2b, k; Biolegend – 116221), TER119-PE (Rat IgG2b, k; Biolegend – 116208); CD105-PE (Rat IgG2a, k; Biolegend – 120407 and CD106-PE (Rat IgG2a, k; Biolegend - 105713) at 1:100 dilution, on ice and in the dark. Cells were washed twice in FACS media and transferred to FACS tubes.

In order to exclude nonviable cells from the analysis, 0.5% of propidium iodide (PI) (P4170, Sigma-Aldrich) was added to the cell suspension 1–2 min prior to analysis. Fifty thousand events (of appropriate size and complexity) per staining were acquired in the cytometer FACS ARIA (BD Biosciences). Subsequent analysis and graphing were performed in the FlowJo VX software.