Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll-Paque (Histopaque; Sigma-Aldrich, UK) density gradient centrifugation. Cells were cultured in RPMI 1640 medium containing 10% FCS and 1% penicillin-streptomycin under normoxia (21% oxygen) or hypoxia conditions (1% oxygen) for 16h, and the supernatants were collected and stored at -80°C until analysis.
Cytometric Bead Array (CBA; BD Biosciences) was used to measure VEGF in cell culture supernatants according to the manufacturer’s instructions. Briefly, beads coated with anti-VEGF antibodies were mixed and incubated with cell culture supernatants or standards for 1h, followed by incubation for 2h with PE-conjugated anti-VEGF detection. Samples were analysed by flow cytometry (FACS CANTO II; BD Biosciences). VEGF concentrations were calculated using the FCAP Array software version 3.0 (BD Biosciences). The total protein concentration was measured using a Pierce BCA protein assay kit (Thermo Fisher Scientific, Renfrew, UK). The concentration of VEGF was normalised to the total protein concentration (pg/mg total protein).
Cytometric Bead Array (CBA; BD Biosciences) was used to measure VEGF in cell culture supernatants according to the manufacturer’s instructions. Briefly, beads coated with anti-VEGF antibodies were mixed and incubated with cell culture supernatants or standards for 1h, followed by incubation for 2h with PE-conjugated anti-VEGF detection. Samples were analysed by flow cytometry (FACS CANTO II; BD Biosciences). VEGF concentrations were calculated using the FCAP Array software version 3.0 (BD Biosciences). The total protein concentration was measured using a Pierce BCA protein assay kit (Thermo Fisher Scientific, Renfrew, UK). The concentration of VEGF was normalised to the total protein concentration (pg/mg total protein).