Cells were then washed with Phosflow Perm/Wash Buffer I (BD Bioscience), cells permeabilized in Phosflow Perm/Wash Buffer I were stained with anti-phosphatidylserine antibody (4B6) or biotin-conjugated anti-PIP3 antibody (Echelon Biosciences Inc).
Following a wash in FACS buffer, cells were stained with anti-GL7 AlexaFluor 647 (eBioscience, UK) at 1 100 dilution, anti-CD95 PE (eBioscience, UK) at 1 100 dilution and anti-B220 PeBiosciencecience, UK) at 1 200 for 30 min at RT in the dark.
After incubation with the blocking buffer, cells were stained with anti-BrdU antibody (1 100; BD Biosciences, Franklin Lakes, NJ, USA) overnight at 4°C.
After washing twice with staining buffer, cells were stained with directly labelled anti-CD3 for 30 min on ice, washed and then fixed with 1% paraformaldehyde.
Cells were washed gently three times with warm buffer and were stained with counterstains as desired amount for imaging.
The slides were washed thoroughly with neutralization buffer and were stained with 10 μL of ethidium bromide (10 μg/mL) and viewed under confocal laser scanning microscopy.
After neutralizing the slides in 0.4 M Tris buffer, they were stained with 40 μl ethidium bromide (2 μg/ml in ultra-pure water).
Following first and secondary antibody incubations in the same buffer, nuclei were stained with blue DAPI (1 μg/ml, Sigma Aldrich).
After washing with Annexin V binding buffer, cells were stained with 2000-fold diluted Cy5-labeled AnnexinV (Biovision, Milpitas, CA) and 500 nM Sytox blue (Invitrogen) and analyzed by flow cytometry.
After further incubation for 18 20 h at 37°C in buffer, gels were stained with Coomassie blue (0.1% [w/v] in 40% [v/v] methanol-10methanol-10%ic aceticolution).
