Arvo mx light wallac 1420 multilabel luminescence counter

The number of viable cells was determined using the Cell Counting Kit-8 (DOJINDO, Kumamoto, Japan) according to the manufacturer’s protocol. Briefly, cells were plated at a density of 5000 cells/well in 96-well plates. After 1–2 h, cells were treated with 10 μM nutlin-3a for 12 h and then 1, 5, 10, 20, 40 or 60 μM cisplatin was added. After 24 h, 10 μL Cell Counting Kit reagent was added to wells, and cells were incubated at 37°C for 3 h. Absorbance was measured at 450 nm using an ARVO MX/Light Wallac 1420 Multilabel/Luminescence Counter (PerkinElmer, Waltham, MA, USA). Cell viability was calculated according to the manufacturer’s protocol for the Cell Counting Kit-8.

To prepare lysates for the measurement of mitochondrial activity, WAT samples were homogenized in homogenization buffer containing 50 mm Tris‐HCl, pH 7.4, 150 mm NaCl, 1% phosphatase inhibitor cocktail (Thermo), 5 mm EDTA, 1% protease inhibitor cocktail (Sigma), 1% NP‐40, and 0.05% sodium deoxycholate. Protein concentration in each lysate was determined using a BCA protein assay kit (Thermo Scientific; IL, USA), according to the manufacturer’s protocol. Citrate synthase (CS) activity was measured by monitoring color development associated with thio‐bis‐(2‐nitrobenzoic) acid (TNB) generation from reduction of DTNB by CoA‐SH, the byproduct of citrate synthesis 24. After addition of reaction buffer, samples were incubated at 25°C for 5 min, and the reaction was initiated by addition of 0.5 mm oxaloacetate. Changes in absorbance at 412 nm were recorded for at least 3 min using an ARVO MX/Light Wallac 1420 Multilabel/Luminescence Counter (PerkinElmer; MA, USA).