Annexin 5 allophycocyanin apc and 7 aminoactinomycin d 7 aad apoptosis detection kit

A flow cytometry-based annexin V allophycocyanin (APC) and 7-aminoactinomycin D (7-AAD) apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, United States) was used to detect apoptotic (annexin V-positive, 7-AAD-negative) and dead (annexin V-positive, 7-AAD-positive) cells. Cell death in annexin V-positive/7-AAD-positive cells may result from apoptosis or necrosis, which can not be distinguished by this assay. For comparisons between transfected cells (and non-transfected cells cultured under the same conditions), cells of different groups were grown and transfected in 6-well plates. 48 h after transfection, the remaining medium of each well was collected and centrifuged to collect the cell debris. Then, cells were washed with PBS and resuspended (together with the corresponding debris collected) in annexin V binding buffer (BD Biosciences), followed by addition of 5 μL APC annexin V and 5 μL 7-AAD reagent to every 10⁵ cells from each sample. After incubation in the dark for 15 min at room temperature, 400 μL binding buffer were added to each sample before analysis by flow cytometry. To assess effects of inhibitors, the procedure was the same, but HTH01-015, WZ4003 or DMSO were added 24 h before flow cytometry.

A flow cytometry-based annexin V allophycocyanin (APC) and 7-aminoactinomycin D (7-AAD) apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, United States) was used to detect cells in apoptosis (annexin V-positive, 7-AAD-negative) and dead cells (annexin V-positive, 7-AAD-positive). WPMY-1 cells were seeded in 6-well plates (250,000 cells/well) and cultured for 24 h. After addition of YM-254890, and DMSO (10 μl/ml) for controls, cells were incubated for 72 h. Subsequently, cells were washed with phosphate-buffered saline (PBS) and resuspended in annexin V binding buffer (BD Biosciences), followed by addition of 5 μl APC annexin V and 5 μl 7-AAD reagent to each sample. After incubation in the dark for 15 min at room temperature, 400 μl binding buffer were added to each sample before analysis by flow cytometry.

A flow-cytometry-based annexin V allophycocyanin (APC) and 7-aminoactinomycin D (7-AAD) apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA) was used to detect apoptotic (annexin V-positive, 7-AAD-negative) and dead (annexin V-positive, 7-AAD-positive) cells. Cell death in annexin V-positive/7-AAD-positive cells may result from apoptosis or necrosis, which cannot be distinguished by this assay. Cells were grown in 6-well plates, and isoflavones or solvent were added 24 h before flow cytometry. The remaining medium of each well was collected and centrifuged to collect the cell debris. Subsequently, the cells were washed with PBS and resuspended (together with the corresponding debris collected) in annexin V binding buffer (BD Biosciences), followed by the addition of 5 μL APC annexin V and 5 μL 7-AAD reagent to every 10⁵ cells from each sample. After incubation in the dark for 15 min at room temperature, 400 μL binding buffer was added to each sample before analysis by flow cytometry.