Annexin 5 propidium iodide

Jurkat human T cell line was kindly provided by Prof. J. Smit (UMCG) and cultured in RPMI (Gibco, 21,875,034) supplemented with 10% FBS, 1 × penicillin–streptomycin, and 1 mM sodium pyruvate. Apoptosis induction for phagocytosis assay (described below) was done on the day of performing the assay as previously described [2 (link)]. Jurkat cells were collected at 1*10⁶ cells/mL in medium and treated with 1 μM staurosporine (Sigma-Aldrich, S5921) for 4 h. To verify induction of apoptosis, Jurkat cells were labelled with Annexin V/propidium iodide (Biolegend, 640,914) according to manufacturer’s instructions and analyzed on a MacsQuant (Miltenyi Biotec). After 4 h of staurosporine exposure, > 90% of cells were early apoptotic (Additional file 7: Fig. S6D).

For resazurin viability assays, 3,000 cells/well were seeded in 96-well plates. The next day, serially diluted inhibitors were added and incubated for 5 days, after which resazurin (Sigma R7017) was added to a final concentration of 0.1 mg/mL and incubated for 5 hours prior to measuring the excitation and/or emission (544/590; Biotek). Cells were reverse transfected (Lipofectamine 3000) with siRNAs according to manufacturer's instructions (Invitrogen) where indicated. To assay for apoptosis, cells were seeded and treated with the indicated inhibitors for 5 days, after which cells were detached using accutase (BioLegend) and stained using Annexin V/propidium iodide following manufacturer's protocol (BioLegend). Cells were kept in the dark until assessment and were analyzed by flow cytometer (BDFACSymphony A3) within 1 hour. To measure cell clonogenicity, 500 cells/well were seeded in a 6-well plate, and treated the following day with the designated inhibitors, and incubated for a total of 19 days with media and inhibitor change every 3–4 days, after which cells were fixed and crystal violet stained. Plates were imaged, destained, and crystal violet quantitated at OD 595 (Biotek).