Biotek synergy mx microplate reader

Enzyme activity assays were performed in cell homogenates for CS, LDH, MDH, and SDH activities and enriched mitochondrial fractions for complex I activity as previously described (56 (link)). Protein concentrations of cell homogenates and enriched mitochondria fractions were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225). The rate of change of absorbance and path length of each well were determined using the BioTek Synergy Mx Microplate Reader (BioTek Instruments, Inc). Enzyme activities were calculated using extinction coefficients of 6.22 mM−1 cm⁻¹ for LDH, MDH, and complex I; 13.6 mM−1 cm⁻¹ for CS; and 19.1 mM−1 cm⁻¹ for SDH.

Quiescent SKOV‐3 cells were pre‐treated with PBSA (10, 50 µmol/L) for 30 minutes, followed by 10% FBS stimulation for 48 hours. In some experiments, quiescent cells were pre‐treated with PBSA or SB203580 for 30 minutes, followed by 10% FBS stimulation for 12 hours. Following stimulation, cells were washed with PBS to remove any residual PBSA or SB203580 and then further stimulated with 10% FBS for another 36 hours until the end of the 48 hours time‐point. After stimulation, conditioned media were collected. VEGF levels in the conditioned media were measured using VEGF ELISA kit (R&D Systems) and analysed at 450 nm with BioTek Synergy Mx microplate reader (BioTek Instruments).24

Cells (10⁶ cells ml^-1; 100 μl per well) were incubated in 96-well microtiter plates and cultured in the presence of the indicated concentrations of F-ara-A. After 48 h of culture, cell viability was assessed by using the kit CellTiter 96© AQUEOUS Assay, following the manufacturer’s instructions. The spectrophotometric absorbance of each sample was measured at 490 nm using the BioTek Synergy Mx microplate reader (BioTek Instruments).