Lt 400 microplate reader

Cells were seeded in 96-well plates at a density of 2–4 × 10³ cells one day before SM or TRAIL treatment. In case of the inhibitor studies, 30 μM necrostatin-1 (nec-1) was included in the treatment, whereas z-VAD-fmk (20 μM) was added 1 h in advance. The cells were incubated for 24–72 h (endpoint measurement) or 2–8 h (time kinetic). Twenty microlitres of MTT reagent (5 mg ml⁻¹) and, after 3 h, 150 μl of solubilisation solution (50% dimethylformamide, 0.2% glacial acetic acid, 20 mM HCl, 10% SDS) was added to each well. After 16–24 h, OD₅₉₅ was measured using the LT-400 microplate reader (Labtech, Uckfield, East Sussex, UK). The statistical analysis of the results was performed using Student's unpaired two-tailed t-test (GraphPad Prism 5 software, La Jolla, CA, USA).