Pe conjugated mouse anti human cd40 antibody

CD40 expression by HCT116 and SW480-CD40 cells was detected using PE-conjugated mouse anti-human CD40 antibody and an isotype control IgG1-PE (BD Biosciences, Berks, UK). In all, 10,000 events were acquired on a Guava EasyCyte flow cytometer and results analysed using EasyCyte software (Luminex, Amsterdam, The Netherlands).

For detection of CD40 expression, cells were cultured in flasks until approximately 80% confluent. Alternatively, for treatment with cytokines, cells were seeded in 24-well plates at 5 × 10⁴ cells/well and, after overnight incubation, they were treated with fresh medium containing 10³ units/ml TNF-α or IFN-γ (R&D Systems/Bio-Techne) for 48 h. Cells were then harvested, washed and re-suspended in FACS buffer^{22 (link)}. CD40 expression was detected using PE-conjugated mouse anti-human CD40 antibody and an isotype control IgG1-PE (BD Biosciences, Berks, UK). In all, 10,000 events were acquired on a Guava EasyCyte flow cytometer and results analysed using EasyCyte software (Millipore, Watford, UK).