Emax microtiter plate reader

Several different methods were used to measure apoptosis and viability. After TRAIL treatment for 5–6 h, cells were stained with Annexin V-Cy5 and propidium iodide (PI), and annexin V⁺ populations were quantified by flow cytometry. After TRAIL treatment for 24 h, cells were stained with PI in hypotonic solution (50 mg/ml PI, 0.1% sodium citrate, 0.1% Triton X-100) overnight at 4 °C. Fractions of cells with sub-G1 DNA content were quantified using CellQUEST software on a FACSCalibur flow cytometer (BD Biosciences). For viability determination, AGS cells were plated in 96-well plates for 24 h before treatment. After TRAIL treatment, the cells were incubated with 0.5 mg/ml MTT in complete medium for 2 h. The surviving cells converted MTT to generate a purple-colored formazan product when dissolved in dimethyl sulfoxide (DMSO). The intensity of formazan product was measured by absorbance at 490 nm using SOFTmax PRO 4.3.1 LS software accompanying an Emax microtiter plate reader (Molecular Device, Sunnyvale, CA). Cell viability was calculated by dividing the absorbance of treated cells by that of the control. For measurement of caspase activation, control and DTX1-expressing cells were treated with 20 ng/ml TRAIL. The levels of procaspase-8, procaspase-3, processed caspase-8 (p43/41, p18) and processed caspase-3 (p19/17) were determined by immunoblots.

Necroptosis was induced by pretreating cells with z-VAD for 0.5 h, followed by stimulation with AT406, BV6, LPS, TNF or IFNβ for the indicated periods. The necroptosis inhibitor Nec-1 was added 0.5 h prior to stimulation for certain experiments. Cell viability of BMDMs or BMDCs was assessed by measuring ATP levels upon adding an equal volume of Cell Titer-Glo reagent (Promega) and incubating for 30 min. Luminescence was determined using a Victor3 1420 Multilabel Counter (PerkinElmer, Shelton, CT). Alternatively, cell viability was determined via reduction of MTT by mitochondrial reductase into purple formazan. The intensity of colored product was measured by absorbance at 490 nm on an Emax microtiter plate reader (Molecular Device, Sunnyvale, CA). Necroptotic HT-29 cells were determined by staining with PI (10 μg/ml) in PBS, and PI⁺ cells were analyzed using flow cytometry. Apoptosis of Jurkat cells was induced by treating with FLAG-FasL and assessed by staining with Annexin V-Cy5 (BD-Biosciences) and subsequent Annexin V⁺ cell quantitation by flow cytometry.