Celltox cytotoxicity assay

CellTox™ cytotoxicity assay (Promega) was used to investigate the cytotoxic effect of DMOG treatment, as the assay measures changes in membrane integrity that occur as a result of cell death. Briefly, cells were seeded in a 96-well plate at a concentration of 500 cells/well and incubated for 48 and 96 h with DMOG (0 mM, 0.01 mM, 0.1 mM, and 1 mM) and tested according to manufacturer's protocol. After each incubation, an equal volume of 100 μL of CellTox buffer containing 1 : 500 dilution CellTox Green Dye was added to each well and incubated at room temperature for 15 minutes. The signal was measured using the VICTOR™ X3 Multilabel Plate Reader with an excitation wavelength of 485–500 nm and emission filter of 520–530 nm. Data were normalized to the untreated control.

Cells were plated in clear-bottomed white walled 96-well plates (Corning) and incubated overnight. Cells were treated with indicated peptides in 1% FBS-containing media for overnight or indicated timings at 37°C. Cell viability was measured with CellTiter-Glo reagent (Promega) according to the manufacturer's instructions. For cytotoxicity assay, cells were plated in black walled 96-well plates (Corning) to allow them to adhere overnight. Upon treatment with a pan-caspase inhibitor, z-VAD-Fmk (100 μM) (Sigma) and subsequent treatment with LTV-tagged peptides, cytotoxicity induced was determined by CellTox cytotoxicity assay (Promega) 30 minutes after addition of peptides according to the manufacturer's instructions. Luminescence was determined using an Infinite 200 multiplate reader (Tecan).