Briefly, the cells were washed three times with PBS, fixed with 4% paraformaldehyde (pH 7.4) for 30 minutes at room Ferrostatin-1 temperature, washed twice, and then permeabilized with 0.1% Triton X (Sigma-Aldrich, St. Louis, MO, USA). After two washes, the cells were incubated with the TUNEL reaction mixture for 60 minutes at 37°C and then washed three times before analysis by confocal microscope (Olympus Fluoview 500, Center Valley, PA, USA). Annexin-V staining Analysis of apoptosis was performed by flow cytometry using Alexa Fluor 488 Annexin-V (Molecular Probes, Invitrogen, USA). 7-AAD (eBioscience,
San Diego, CA, USA) was used for the discrimination of dead cells. Briefly, the cells were dissociated with 0.025% trypsin and 0.01% EDTA, washed two times with PBS and incubated in 100 μl annexin-binding PF-01367338 chemical structure buffer containing 5 μl Alexa Fluor 488 Annexin-V for 15 minutes at room temperature. After washing in PBS, the samples were resuspended in 300 μl of annexin-binding buffer containing 5 μl 7-AAD and analyzed by flow cytometry using a FACSCalibur System
(BD Biosciences, San Jose, CA, USA). Quantitative PCR Array A focused panel of 86 apoptosis-related genes (qPCR-Array) was customized by SuperArray (Bioscience Corporation, Frederick, MD, USA) on a 96 well format including endogenous controls. The qPCR-Array was optimized for template and PCR conditions according to the manufacturer’s recommendations. The total RNA was isolated and purified as described previously [29] and first strand cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA) according to the manufacturer’s instructions. The real time PCR reaction cocktail
was prepared by mixing 1125 μl of 2× SuperArray PCR master mix (RT2 Real-Time™ SYBR Green/ROX (Cat. No. PA 012), 2 μg of cDNA, and 1127 μl of ddH2O. The final volume was adjusted to 2450 μl and 25 μl of the cocktail was loaded onto each well. 10 fold serial dilutions of experimental cocktail were used for β-actin gene to check the linearity and consistent amplification across the panels. The plate was loaded on to ABI 7500 over Real Time PCR machine (Applied Biosystems, Foster City, CA, USA) and the reaction was carried out using relative quantification method with the following conditions: 1 cycle at 95°C for 10 minutes followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. The dissociation curve was drawn up after completing the relative quantification method which ensures specific amplification. The PCR-Array was duplicated for each sample and fold differences were calculated according to the ΔΔCt method using GAPDH as the endogenous control. Statistical analysis All data are expressed as the mean ± SD.