Fitc conjugated anti cd31

The HUVECs were isolated using a standard collagenase digestion method [19 (link)] as we do routinely in our laboratory [20 (link)]. Immediately after isolation, the cells were cultured and expanded in endothelial cell medium (ECM cat number 1001, ScienCell, San Diego, CA) containing 5% fetal bovine serum, 1% penicillin/streptomycin, and endothelial cell growth supplement in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. For this study, 80% of confluent HUVEC monolayers (passages 2-3) were used.
The purity of primary HUVEC cultures was evaluated by flow cytometry analysis performed on Guava EasyCyte8. Briefly, detached cells were resuspended in 200 μL of PBS containing 1% bovine serum albumin (Sigma-Aldrich, Saint Luis, MO, USA) and incubated for 15 min at 20°C with the following antibodies (Ab): FITC-conjugated anti-CD31, PE-A-conjugated anti-CD144, PE-Cy7-A-conjugated anti-CD146 (BioLegend, San Diego, CA, USA), PE-A-conjugated anti-CD105 (Bioscience Pharmingen, San Jose, CA, USA), and APC-A-conjugated anti-CD45 (DAKO, Santa Clara, CA, USA). Data files were collected and analysed using the FACSDiva software program (version 6.1.3; BD Bioscience, San Jose, CA, USA).

Femurs of wild-type mice were collected and incubated for 30 min at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% collagenase D (from Clostridium histolyticum, Sigma-Aldrich, catalogue no. C5138-1G), 1 U ml⁻¹ of dispase (Thermo Fisher Scientific, catalogue no. 17105-041) and 1 U ml⁻¹ of DNase (Invitrogen) before cells were dissociated by trituration. Cells were dissociated to generate a single-cell suspension by filtering through a 40-μm nylon mesh. RBCs were eliminated using RBC Lysis Buffer (pluriSelect, Deutscher, catalogue no. 60-00050-11). Cells were subjected to Fc blocking (BioLegend, catalogue no. 101320, 1:200) and stained with FITC-conjugated anti-CD31 (BioLegend, catalogue no. 102506, 1:200), allophycocyanin (APC)-conjugated anti-CD45 (BioLegend, catalogue no. 103112, 1:200), phycoerythrin (PE)-conjugated anti-Ly6a (BioLegend, catalogue no. 122507, 1:200) and APC/Cy7-conjugated anti-Ly6c (BioLegend, catalogue no. 128025, 1:200) antibodies for 40 min. Analysis was performed with an SH800S Cell Sorter (SONY) and FlowJo software (Tree Star).