Annexin v 7 aad apoptosis detection kit

Cells were seeded in 96-well plates at a density of 2,000 cells per well. The growth rate of cells was evaluated using the CCK-8 cell proliferation kit (Dojindo Laboratories, Kumamoto, Japan), according to the manufacturers’ instructions. OD detection at 450 nm was carried out by infinite 200Pro (Tecan). Cell apoptosis was analyzed using the Annexin V/7-AAD Apoptosis Detection kit (Keygen Biotech, Nanjing, China) on a CytoFLEX flow cytometer (Beckman, California, United States). Results were further analyzed by Flowjo 10.4.

Cells were seeded in 96-well plates at a density of 1000 cells per well. The growth rate of cells was evaluated by using the CCK-8 cell proliferation kit (Dojindo Laboratories, Kumamoto, Japan), according to the manufacturers’ instructions. Cell-cycle analysis was carried out by flow cytometry (Beckman CytoFLEX, California, USA) after propidium iodide staining.
For analysis the role of SH3BGRL in anticancer drug-induced apoptosis, parental MCF-7 and SH3BGRL-overexpressing MCF-7 cells were treated with 10 μM Cisplatin (TOCRIS, Abingdon, OX, UK) for 12 h or with 150 ng Herceptin (Roche, Basel, Switzerland) for 96 h, respectively. Likely, MDA-MB-453 and MDA-MB-453 SH3BGRL knockdown cells were treated with 10 μM Cisplatin for 12 h, with 150 ng Herceptin for 96 h, PI3K/AKT inhibitor LY294002 (CST, Danvers, MA,USA) for 24 h, or Herceptin and ATK inhibitor combination for 48 h, respectively. Cell apoptosis was analyzed using the Annexin V/7-AAD Apoptosis Detection kit (Keygen Biotech, Nanjing, China), and the percentage of apoptotic cells was analyzed by flow cytometer (Beckman CytoFLEX, California, USA). The detailed procedures of all above assays were described as previously [16 (link)].

The apoptosis assay was conducted using the Annexin V-7AAD Apoptosis Detection Kit (Keygen Biotech) according to manufacturer’s instructions. The cells were then analyzed using FACS flow cytometry (BD Biosciences Inc.)

The cells were trypsinized and centrifuged at 1500 rpm for 3 min. After removing the supernatant, the cells were washed with PBS and resuspended. An aliquot containing 1 × 10⁵ cells was collected and mixed with 500 μl of the binding buffer. Then, 5 μl of annexin V and 5 μl of 7‐aminoactinomycin D (annexin V/7‐AAD Apoptosis Detection Kit, KeyGen, Nanjing, China) were added to the cell suspension. After a 15‐min incubation, the suspension was cooled on ice and analysed by flow cytometry.

Cells were cultured in serum-free medium for 48h. Then, the apoptosis assay was done with the Annexin V-7AAD apoptosis detection kit (Keygen Biotech) according to the manufacturer's instructions. Cells were then analyzed by FACS cytometry (BD Biosciences Inc.).