Apoptosis Detection in Cell Samples

Samples of GSC and RSC (1 ml suspension) were collected at day 0 and 6 and washed three times in phosphate buffered saline (PBS), incubated with fixation solution of 2 % (v/v) paraformaldehyde in PBS for 60 min. Then, samples were treated with permeabilisation solution (0.1 % (w/v) Triton X-100 in 0.1 % (w/v) sodium citrate) and washed three times with PBS. Samples were labelled with TUNEL reaction mixture (TMR-red in situ cell death detection kit, Roche Diagnostics) in darkness at 37 °C for 60 min. Negative (without terminal transferase) and positive (with ethanol treatment at day 0, as described by Hogg et al. [33 (link)]) samples were properly included. For nuclear staining, the samples were washed twice by PBS and stained with 1 μg ml⁻¹ 4′,6-diamidino-2-phenylindole (DAPI) for 15 min. Finally, all samples were examined under a Leitz Fluovert fluorescence microscope, with two sets of filters: 360 nm excitation and 420 nm emission for DAPI detection; 540 nm excitation and 620 nm emission for TUNEL, respectively. The percentage of apoptotic TUNEL-positive nuclei was determined by counting at least 200 nuclei.