Cytokine bead array

In order to generate an ID8Luc CCL5 knockdown cell line, the lentiviral vector pLKO.1-puro was used. For the propagation of retroviral particles 293T cells were seeded at 6 × 10⁶ per T75 tissue culture flask in RPMI-10+10% FBS medium 24 hr before transfection. Cells were then transfected with 10 µg pLKO.1-puro CCL5 short hairpin plasmid, 1.58 µg pCMV-PAX2 plasmid and 3.125 µg of pMD2G plasmid using 59.175 µl Turbofect (Fisher Scientific). The viral supernatant was harvested at 24 and 48 hr post-transfection. PLKO.1-puromycin non-specific targeting lentiviral particles were also produced and used as control. The transduction of ID8Luc cells with lentiviruses was carried out as described above. The infected cells were then selected with puromycin (2 µg/ml). Secretion of CCL5 was assessed using the BD cytokine bead array as per manufacturer’s instructions. Luciferase expression was also assessed post retroviral infection and ID8Luc CCL5^KD expressed luciferase 1.4-fold higher than ID8Luc scr sh cells (data not shown).

Cytokines concentrations were determined in cell-free supernatants of 48-hour cell cultures using the BD cytokine bead array according to manufacturer’s recommendations. All Flex Sets are listed in the Key resource table.
ID8 cells (1 × 10⁵) were seeded and cultured in 6-well plates and treated with Olaparib (10 μM) for 48 h. Quantitative determination of VEGF-A in cell culture supernatants was assessed using a Mouse VEGF-A ELISA Kit according to the manufacturer’s instructions and after cell number normalization.