Caspglow fluorescein active caspase staining kit

Apoptosis was measured using the Thermo Fisher Scientific CaspGLOW Fluorescein Active Caspase Staining Kit. Immediately following cell isolation, splenocytes were incubated with Z-VAD-FMK-FITC for 45 min at 37°C. Cells were washed according to the manufacturer’s instructions and stained with a fixable viability dye and antibodies against surface antigens as described above.

Multicolor flow cytometry for analysis or for sorting was performed on an LSR Fortessa-5 or FACS Aria (BD Biosciences), respectively. Single-cell suspensions of splenocytes were blocked with anti-CD16/32 mAb (clone 2.4G2), followed by staining with the following antibodies: anti-B220 (clone RA3-6B2) and anti-CD45.2 (clone 104) from BD Biosciences. HEL-binding B cells were stained as described previously (Chan et al, 2009 (link)). For cell cycle analyses, spleen cells were first stained for extracellular antigens and then were analysed with 10 μg/ml DAPI staining using a Cytofix/Cytoperm kit (BD Biosciences) or PFA and Tween-20. Cell cycle was calculated by FlowJo Dean/Jett/Fox algorithm or by setting gates manually. The Click-iT EdU Alexa Fluor 488 Imaging kit and CaspGLOW Fluorescein Active Caspase Staining kit (both from Thermo Fisher Scientific) were used according to the manufacturer’s instructions. Data were analysed with FlowJo software (Tree Star).