Apo brdu tunel assay

The APO-BrdU TUNEL Assay (Invitrogen Cat. A23210) was used according to the manufacturer’s instructions and applied to whole hSCO. Incubation times were modified to adjust for tissue size. hSCOs were incubated in 70% EtOH for at least 24 h prior to additional processing. The DNA-labeling step was allowed to proceed for 24 h at 37°C. Antibody labeling steps were conducted using the IF strategies described above.

Apoptosis and cell cycle distribution were investigated using flow cytometry. For apoptosis, APO-BRDU (TUNEL) assay (Life Technologies) was performed, according to the manufacturer’s instructions. For investigation of cell cycle distribution, 2*10⁶ cells were harvested and resuspended in 200 μl ice-cold PBS and added to 4 ml ice-cold ethanol and incubated on ice for 45 min. Then, 6 ml of ice-cold staining buffer (SB: 0.5% BSA in PBS) was added, and the cells were centrifuged at 300 × g, at 4°C for 5 min. The pellet was resuspended in 1 ml SB and the centrifugation repeated. The cells were resuspended in 300 μl SB containing 2 μg/ml Hoechst 33342 (Sigma-Aldrich). For both assays, the LSR II UV Laser (BD Bioscience) was used, and the data was analyzed using FlowJo v8.8.7 software (Tree Star).

In order to determine effects on cell proliferation and apoptosis by 5-bromo-2'-deoxyuridine (BrdU) incorporation, 4',6-diamidino-2-phenylindole (DAPI)-staining and Apo-BrdU TUNEL assay (Life Technologies), 1 x 10⁵ WERI-Rb1 cells, synchronized by serum-starvation, were seeded on cover slips and cultivated in 24-well plates in 300 μl medium containing 5% FCS in the presence of single additives or treated with a combination of several substances as summarized in Table 1. Medium without insulin was used as insulin has been shown to interfere with apoptosis induction (personal observation).
For the determination of effects on cell proliferation, BrdU (5 μM; Sigma) was added 6 h before the end of culture time and BrdU immunocytochemistry was performed as described previously [20 (link)]. BrdU positive cells and 4',6-diamidino-2-phenylindole (DAPI)-stained pycnotic nuclei were counted microscopically. For this purpose, at least 10 different fields of view of one coverslip and at least 1000 cells were counted and the number of DAPI-positive, clearly pycnotic cells with condensed nuclei or obvious apoptotic bodies (at least 10) or clearly BrdU-positive stained cells was determined.

Cell proliferation was determined by 5-Bromo-2´-deoxyuridine (BrdU; Sigma) incorporation. For BrdU immunocytochemistry 10 μM BrdU was added to the cells 4 h prior to PFA fixation. Cells were incubated with a rat anti-BrdU antibody (1:1,000; ab6326; Abcam) and proliferating cells were visualized using a goat anti-rat antibody labelled with Alexa Flour^® 594 (1:1,000; Molecular Probes). In order to determine changes in apoptosis levels, cells were stained with 4',6-Diamidino-2-phenylindole (DAPI; Sigma) or Apo-BrdU TUNEL assay (Life Technologies), following the manufacturer`s protocol and pycnotic nuclei were counted manually as described previously by our group [27 (link)].