Annexin 5 cy5 reagent

Co-cultures were carried out in flat bottom 96-well-plates. For IFN-γ secretion tests T cells and targets were plated 100,000 cell/well each overnight in target cell medium. ELISA for the detection of human IFN-γ was carried out using Biolegend's Elisa MAX™ Deluxe kit. For cytotoxic assays, targets were stained by CellTrace™ Violet according to manufacturer's instructions and seeded 40,000 cells/well. T cells were then added at 160,000 cells/ml. After 2.5–3 h at 37°C, cells were collected from wells and stained for apoptosis by Annexin V-Cy5 reagent (Biovision). CellTrace positive cells were assessed for AnnexinV staining by flow cytometry.

6 well plates were seeded with 1×10^5 cells and after a 24 hour recovery period were treated as described in results and then harvested with trypsin 0.05%. After washing and re-suspending cells in 200uL of PBS, 1 μL of AnnexinV-Cy5reagent (Biovision Inc.) and 1 μL of PI (1 mg/mL) were added and cells were incubated in dark for 15 minutes on ice then immediately analyzed on a Stratedigm S1400 flow cytometer (Stratedigm Inc.). Samples were analyzed by drawing quadrants around the healthy cell population in the negative control samples using FlowJo software. Apoptotic cells were taken to be the percentage of Annexin V positive cells. All measurements were repeated in at least 3 independent experiments.