7 aminoactinomycin d viability dye

Adherent cells were detached from tissue culture plates using trypsin-EDTA, washed with PBS, and resuspended at 1 × 10⁶ cells/mL in Hank’s balanced salt solution (HBSS; Mediatech^®) with 0.5% w/v bovine serum albumin (BSA; Amresco^®, Solon, OH, USA) and 2 µg/mL Fcγ fragment of goat anti-human IgG antibody. After incubation at 4 °C for 30 min, a primary antibody (details below) was added to the cell suspension (100 µL) which was then incubated for another 30 min at 4 °C. If the antibody did not have a fluorophore, cells were spun down at 450 g for 2 min, resuspended to 100 µl HBSS with 0.5% w/v BSA, and incubated with a fluorophore-conjugated secondary antibody (details below) at 4 °C for 30 min. Cells were finally spun down at 450× g for 2 min and resuspended in 200 µL HBSS with 0.5% w/v BSA for flow cytometry on a FACSCalibur™ machine (BD Biosciences^®). Intensities for forward and side scatter, and fluorescence in the FL1, FL2, and FL4 channels were recorded for 10–20 thousand events with CellQuest™ Pro software (version 5; BD Biosciences^®). The 7-amino-actinomycin D viability dye (Sigma^®) was added to the cell suspension at 1 µg/mL immediately before cytometry. Cytometry data were analyzed with FCS Express™ 4 (De Novo Software^®, Pasadena, CA, USA).