Annexin 5 apc apoptosis kit

Apoptosis detection by flow cytometry: When HCCC-9810 and HUCCT1 cells were grown to 85% coverage, they were harvested after trypsinization. Cells were washed once with pre-chilled D-Hanks and 1 × binding buffer, respectively. The cells were resuspended in 200 µL of 1 × binding buffer, and then incubated with 10 µL of Annexin V-APC (apoptosis kit, eBioscience) for 15 min at room temperature in the dark. Finally, the level of apoptosis was detected by flow cytometry (Millipore).
Cell cycle detection by flow cytometry: When the coverage of HCCC-9810 and HUCCT1 cells exceeded 70%, cells were harvested after trypsinization. After washing once with pre-cooled PBS, cells were fixed in 70% ethanol pre-cooled at 4 °C for 1 h. After washing with PBS, the cells were resuspended with the prepared staining solution, and the cell cycle was detected by flow cytometry. Preparation ratio of cell staining solution: 40 × PI solution (2 mg/ml, Sigma): 100 × RNase solution (10 mg/ml, TakaRa): 1 × PBS = 25:10:1000.

In apoptosis assays, cultured erythroid cells were washed and labeled using the annexin V- eFluor 450 apoptosis kit, the annexin V-APC apoptosis kit or the annexin V-PE apoptosis kit (eBioscience), according to the manufacturer’s recommendations. For flow cytometry differentiation analyses, cells were washed with PBS at the indicated time and then stained for KIT/GPA expression with 1:30 human APC-anti-CD117 (eBioscience), 1:10 human PE-anti-235a (Beckman Coulter),1:20 human APC-anti-235a (Miltenyi Biotec) or 1:20 human Vioblue-anti-235a (Miltenyi Biotec). Live GFP + or CFP + cells were analyzed. For CD49d/Band3 expression, cells were stained with 1:100 human PE-BRIC6 (IBGRL Research) and 1:20 human APC-anti-CD49d (Beckman Coulter). Cells were washed with PBS and labeled with the annexin V-eFluor 450 apoptosis kit, and GFP + /annexin V- cells were analyzed for differentiation markers. FACS experiments were conducted on a Beckman Coulter Gallios or BD Fortessa Flow cytometer. The data were analyzed using FlowJo software (version 10.0.8, TreeStar)