In situ cell death detection kit 1

Cells were fixed in fresh 4% paraformaldehyde and 4% sucrose in PBS for 20 min at room temperature and permeablized in 0.1% Triton X-100 and 0.1% sodium citrate in PBS for 2 min on ice. Terminal deoxynucleotidyl transferase-biotin dUTP nick-end labeling (TUNEL) staining was performed using the in situ cell death detection kit I as described by the manufacturer (Roche, Basel, Switzerland). The coverslips were washed once in PBS for 5 min and then mounted on glass slides to be observed under a fluorescence microscope. Data were expressed as the ratio of apoptotic cells to total cells.

Neurons seeded in 96-well plates that were subjected to cell viability assay were incubated with 0.5 mg/mL MTT for 2 h. After discarding the medium, DMSO was added to dissolve the formazan crystals that yield by MTT. The absorption of the solution was measured at 490nm by Epoch 2 microplate spectrophotometer (BioTek Instruments, Inc.).
TUNEL experiments probing death cells were carried out by measuring the 3'-OH of DNA strand breaks. Cell was fixed with 4% paraformaldehyde for 15 min at room temperature, then permeabilized with 0.1% Triton X-100 and 0.1% sodium citrate in 1x PBS for 3 min on ice. TUNEL assay was performed with in situ cell death detection kit I (#Roche-11684795910). Cell death rate was obtained from the ratio of TUNEL-positive cells vs DAPI positive cells.