Chemidoc1 imaging system

TissueLyser LT (Qiagen, Hilden, Germany) was used to prepare protein extracts. Protein concentrations of tissues lysates were determined using the BCA Protein Assay (Thermo Scientific). The levels of multiple apoptosis protein were examined by the Human Apoptosis Antibody Array - Membrane (ab134001, Life Technologies), prepared for the simultaneous detection of 43 proteins. The following targets could be detected by this array.
Three samples from each group were diluted to a final concentration of 5 μg/μL. The cytokine array was performed according to the manufacturer’s instructions. Chemiluminescence detection was performed using multiple exposure times (30 s to 5 min) with the ChemiDoc1 Imaging System using Quantity One Basic Software (Bio-Rad, Hercules, CA, USA).

The expression of selected cytokines was measured using cytokine arrays, which are antibody-pair-based assays that are analogous to Western blots, but using a membrane as a substrate.
The following groups were created:
I. 100 µg/mL of extract with H₂O₂II. H₂O₂ alone
III. Control –PBS only.
Cells at 75 cm³ flask in complete medium as described in section ‘Cell cultures and treatments’, except for the FBS concentration, which was reduced to 1%. After 24 h, extract (100 µg/mL in PBS) or an equivalent volume of PBS was added to cells. Following a 72 h incubation period, oxidative stress was induced by adding 100 µM H₂O₂. Cell pellets from individual groups were pooled to obtain 3 samples: 100 µg/mL extract + H₂O₂, H₂O₂ alone, and the control group. Total protein was isolated and measured using the BCA method. The isolated protein was analyzed using the human cytokine antibody array (Abcam), according to the manufacturer’s instructions. he same amount of protein was applied to each membrane. Chemiluminescence detection was performed using the ChemiDoc1 Imaging System and Quantity One Basic Software (Bio-Rad, Hercules, CA, USA). The captured images of each membrane were analysed using Fiji software (National Institute of Health, Bethesda, MD, USA). The results from each group were optimized according to the Abcam protocol (N = 4).