Quantitative caspase 3 elisa kit

To assess the mechanism of VK2 on apoptosis, the active caspase-3 level was measured by using quantitative caspase-3 ELISA kit (R&D Systems, Inc. USA). Cells were treated with various concentrations of VK2 (25, 50, 100, 150 and 200μM) and drug free media (untreated) for 48hrs. After incubation, cell extracts were prepared according to manufacturer’s instructions. Cells were mixed with lysis buffer and cell lysates were transferred into the wells of a microplate pre-coated with a monoclonal antibody specific for caspase-3. Substrate solution (Streptavidin-HRP) was added to the wells. The enzyme reaction yielded a blue product that turned yellow when a stop solution was added. Optical density was determined within 30min using a microplate reader set to 450nm with a wavelength correction at 540 nm or 570nm. The active caspase-3 concentrations were calculated from a standard curve constructed with known concentrations of active caspase-3. Caspase concentration was expressed as ng/mL protein.

The active caspase-3 level was measured using quantitative caspase-3 ELISA kit (R&D Systems, Inc. USA). Cells were treated with various concentrations of DRP-27 and vehicle alone (control group) for 48 h. After incubation, cell extracts were prepared according to manufacturer instructions. Briefly, cells were mixed with the lysis buffer and cell lysates were transferred into the wells of a microplate, pre-coated with a monoclonal antibody specific for caspase-3. Following this, substrate solution (streptavidin-HRP) was added to the wells. The enzyme reaction yielded a blue product that turned yellow when a stop solution was added. The optical density of each well was determined within 30 min, using a microplate reader (Synergy 2 Multi-Mode Reader, BioTeK, Winooski, Vermont, USA) set to 450 nm with a wavelength correction at 540 nm or 570 nm. The active caspase-3 concentrations were calculated from a standard curve constructed using known concentrations of active caspase-3.

To assess the effect of Nef on apoptosis, the active caspase-3 level was measured by using quantitative caspase-3 ELISA kit (R&D Systems, Inc. USA). The protocol was followed according to Pan et al., 2016 (link) with some modifications. In brief, cells were treated with various concentrations of Nef (10, 25, 50μM) and drug free media (control group) for 48 hrs. After incubation, cell extracts were prepared according to manufacturer’s instructions. Cells were mixed with lysis buffer and cell lysates were transferred into the wells of a micro-plate pre-coated with a monoclonal antibody specific for caspase-3. Substrate solution (Streptavidin-HRP) was added to the wells. The enzyme reaction yielded a blue product that turned yellow when a stop solution was added. Optical density was determined within 30 minutes using a micro-plate reader set to 450nm with a wavelength correction at 540nm or 570nm. The active caspase-3 concentrations were calculated from a standard curve constructed with known concentrations of active caspase-3.