Kaluza flow cytometry analysis software version

EDTA detached HUVECs were washed twice with PBS containing 1 mM EDTA and 2% FBS; and then stained with α-CD130 (gp130)-APC (clone: 2E1B02) and α-CD126 (IL-6Rα)-PE (clone: UV4) antibodies (BioLegend, UK) for 25 min at 4 °C in the dark. Fluorescence minus one (FMO) controls were used as negative control for staining. 7AAD staining was performed to remove dead cells from analyses. Stained cells were acquired using Gallios™ Flow Cytometer (Beckman Coulter Life Sciences, UK) and analyzed using Kaluza flow cytometry analysis software version 1.3 (Beckman Coulter, UK).

After respective treatments, media (including any floating cells) and all remaining cells were collected. 2×10⁵ cells/sample were fixed with 70% ice-cold ethanol for 2 min while vortexing. Samples were centrifuged at 2,500 rpm for 5 min at 4°C. The cell pellet was resuspended in 200 μL of PBS, RNAse A at a final concentration of 30 ng/mL (Sigma-Aldrich) was added and samples were cultivated for 30 min at 37°C. PI at a final concentration of 1 μg/mL was added to the samples 15 min prior analysis by flow cytometry (Beckman-Coulter). Results were analyzed with Beckman Coulter's Kaluza flow cytometry analysis software (version 1.1).