Apo direct kit

Nuclear fragmentation in fixed cells (4% paraformaldehyde/phosphate-buffered saline) was detected via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining using an APO-DIRECT™ Kit (Phoenix Flow Systems, San Diego, CA, USA) according to the manufacturer’s protocol; DAPI was used to counterstain the nuclei. Apoptosis-positive cells and DAPI-positive cells in four rectangular areas (0.75 × 1.0 mm) were counted on each well, and their average values were calculated. The cells were manually counted by two blinded investigators. The rate of apoptosis-positive cells (number of apoptosis-positive nuclei/number of DAPI-positive nuclei) was determined as the mean value of the four areas (the replicate number was 3, n = 10 per group).

According to previous studies [18 (link), 19 (link)], nuclear fragmentation was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining with an APO-DIRECT Kit (Phoenix Flow Systems, San Diego, CA, USA) according to the manufacturer’s protocol, using fixed cells (4 % paraformaldehyde/PBS) with 2-(4-amidinophenyl)-1 H-indole-6-carboxamidine (DAPI). For quantification, the number of apoptosis-positive and DAPI-positive cells in four rectangular areas (0.75 mm × 1.0 mm) in each slide was counted, and the mean values were calculated. Each area was randomly selected and the cells were manually counted by two blinded investigators. The percentage of apoptosis-positive cells was calculated using the formula (number of apoptosis-positive nuclei/number of DAPI-positive nuclei) × 100 and expressed as a mean of the four areas (n = 15 per group).

The Bax, PERK, IRE1α, BiP, CHOP and β-actin primary antibodies were obtained from Cell Signaling Technology. The androgen receptor (AR) and caspase-4 primary antibody and the anti-mouse and anti-rabbit HRP conjugated secondary antibodies were obtained from Santa Cruz Biotechnology, Inc. Anti-mouse and anti-rabbit fluorescent labeled secondary antibodies were obtained from Invitrogen. CHOP siRNA, BiP siRNA and control siRNA were purchased from Santa Cruz Biotechnology, Inc. and the TransIT-siQuest reagent from Mirus Bio LLC. The APO-DIRECT Kit was obtained from Phoenix Flow Systems, BrdU Cell Proliferation Assay Kit and PathScan Cleaved Caspase-3 Sandwich ELISA Kit from Cell Signaling Technology, DeadEnd Fluorometric TUNEL System from Promega and the PSA ELISA kit from Anogen.

Nuclear fragmentation was detected by TUNEL staining with an APO-DIRECT™ kit (Phoenix Flow Systems, San Diego, CA, USA) using fixed cells (4% paraformaldehyde/PBS) and DAPI. For quantitative analysis of apoptosis, the number of apoptotic cells and DAPI-positive cells in four randomly selected areas on each slide was counted by two independent investigators, and the mean value of each was calculated. The percentage of apoptotic cells was calculated using the following formula: (number of apoptosis-positive cells/number of DAPI-positive cells) × 100 (n = 8 per group).