Annexin 5 cy5 apoptosis detection kit

Infected cells were subjected to Annexin V staining using AnnexinV-Cy5 apoptosis detection kit from Abcam (Cat. No. ab14150) according to manufacturer's protocol. Cells were counterstained with propidium iodide and analyzed for apoptosis by fluorescence activated cell sorting (FACS) using a BD LSRII analyzer and Flowjo software.

Metabolic MTT (3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide) conversion by viable cells was used to monitor cell expansion as described^{5 (link)}. At day 0, 10,000 cells were seeded per well of a 96-well plate in triplicates and analysed after the indicated times. Apoptosis was assayed by staining of the cells with propidium iodide (PI) and Cy5-labeled annexin V using the Annexin V-Cy5 Apoptosis Detection Kit (K103, Biovision) as described in the manufacturer’s protocol. Flow cytometric analysis of PI and annexin V-Cy5 staining was performed with the FACS CantoII flow cytometer (Becton Dickinson). FACS data were analysed with FlowJo X.0.7 software. The gating strategy for apoptosis assays is shown in Supplementary Fig. 8. All cells were included into the analysis, whereas cell debris was excluded.

Streptavidin (Jackson) was conjugated with biotinylated Ro09–0198 (provided by Dr. M. Umeda, Kyoto Univ., Japan) and separated from free biotinylated Ro09–0198 using a Sephadex G-25 gel filtration column (GE Healthcare). For staining of cell surface PE, cells were incubated with 50 μg/ml SA-Bio-Ro for 30 min in α-MEM containing 0.2% (w/v) fatty acid-free bovine serum albumin (BSA) (Sigma). The cells were washed with 20 mM HEPES, pH 7.4, 115 mM NaCl, 5.4 mM KCl, 2.2 mM CaCl₂, 0.8 mM MgCl₂ and 13.8 mM glucose, and then fixed with 4% (w/v) paraformaldehyde in PBS for 30 min, followed by a 5-min permeabilisation with 0.1% (v/v) Triton X-100 in PBS. Immunostaining was performed with a FITC-conjugated anti-streptavidin antibody (Vector Laboratories). F-actin was stained using Alexa Fluor 594-conjugated phalloidin (Molecular Probes). PS on the cell surface was detected with an annexin V-Cy5 apoptosis detection kit (BioVision) in accordance with the manufacturer’s protocol. Fluorescence microscopy was performed using a DSU-IX81 microscope (Olympus). The images were photographed with a C10600 digital camera (Hamamatsu Photonics). In some experiments, immunofluorescence intensity of cells cultured in 96-well plates was quantified with an ImageXpress Micro XL HCS (Molecular Devices) and MetaXpress Image Analysis software (Molecular Devices).