Synergy h4 fluorescence microplate reader

Caspase activity was determined by Colorimetric Protease Assay Kits (Invitrogen, Thermo Fisher Scientific Inc., MA, USA), whereas calpain activity was measured by Fluorometric Assay Kit (ab65308, Abcam, Cambridge, UK). Briefly, the IC₅₀ of GTN and z-VAD-fmk-treated cell pellets were lysed with RIPA lysis buffer on ice, and an equal amount of proteins from each cell pellet was prepared. Caspase-8 (Ile-Glu-Thr-Asp, IETD)-pNA), caspase-3 (Asp-Glu-Val-Asp, DEVD-pNA), caspase-9 (Leu-Glu-His-Asp, LEHD-pNA) chromogenic substrates and calpain ac-Leu-Leu-Tyr-7-amino-trifluoromethyl coumarin (ac-LLY-AFC) fluorogenic substrate were added for 1 h at 37 °C. Caspase activity as optical density was measured at a wavelength of 405 nm by a spectrophotometric microplate reader. Calpain activity was determined by a Synergy™ H4 fluorescence microplate reader (BioTek, Winooski, VT, USA) at a wavelength of 400/505 nm (excitation/emission).

MDA-MB-231 cells were simultaneously treated with GTN at various concentrations and combined with or without 10 µM of z-VAD-fmk for an hour. The cells were then added to 40 μM 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) and 20 μM dihydroethidium (DHE) to detect hydrogen peroxide and superoxide anion radicals, respectively. After 30 min of incubation, fluorescence intensity was measured by a Synergy™ H4 Fluorescence Microplate Reader (BioTek, Winooski, VT, USA) at excitation/emission wavelengths 485/530 nm and 535/635 nm for DCF and DHE, respectively.