Optima software 2

IL6, IL8, IL11, IL15, and VEGF concentrations in cell culture supernatant of RCC cells and nonmalignant RC-124 cells were determined by use of target-specific DuoSet ELISA kits with a Substrate Reagent Pack containing stabilized hydrogen peroxide and tetramethylbenzidine (all R&D, Minneapolis, MN, USA) according to the supplier's instructions. Cells were sedimented (1.3 × g, 5 min), the supernatant was incubated with capture antibodies (overnight, 4°C), and soluble antigens were determined applying the BMG FLUOstar OPTIMA Microplate Reader with OPTIMA software 2.10 (BMG Labtech, Offenbach, Germany). Each sample was analyzed in duplicates.

HSP27 concentration in cell culture supernatant was determined by use of the DuoSet ELISA kit human HSP27 (DY1580; R&D, Minneapolis, MN, USA) with Substrate Reagent Pack containing stabilized hydrogen peroxide (Part #895000; R&D) and stabilized tetramethylbenzidine (Part #895001; R&D) according to the manufacturer's instructions. Duplicates of each sample were measured. Cells and cellular debris were sedimented (1, 3 ×g, 5 min), the supernatant was incubated with capture antibodies (overnight, 4°C), and soluble HSP27 was determined using a BMG FLUOstar OPTIMA Microplate Reader with OPTIMA software 2.10 (BMG Labtech, Offenbach, Germany).