Micro bicinchoninic acid assay bca protein assay kit

Multi-apoptotic protein detection arrays (Human Apoptosis Antibody Array Kit; R&D Systems, Inc.) were used to determine possible apoptotic pathways involved in cell death according to the manufacturer’s instructions. Cells were treated with 0 mg/ml, 1 mg/ml, 50 mg/ml, or 100 mg/ml TXA for one hour. The protein content of each well was quantified using a Micro bicinchoninic acid assay (BCA) Protein Assay Kit (Thermo Fisher Scientific) and read on a microplate reader at 540 nm. Lysate protein concentrations were then normalized in lysis buffer. Cells were cultured in 12-well plates for one hour, with increasing concentrations of TXA with medium (0 mg/ml, 1 mg/ml, 50 mg/ml, or 100 mg/ml). Cell supernatants were aspirated and discarded. After blocking of non-specific binding at room temperature for one hour, antibody array membranes were incubated with the cell lysate (1.5 ml) at 4°C overnight and then with a diluted solution of horseradish peroxidase (HRP)-conjugated streptavidin at room temperature for 30 minutes. Visualization of protein expression was carried out by chemiluminescence and signal intensity was quantified using Luminex (Azure c500; Azure Biosystems, Dublin, California). The immunoreactive band density was analyzed using WinImage studio version 5.2 (LI-COR Biosciences, Lincoln, Nebraska).