Annexin 5 apc 7 aad apoptosis kit

We used the Cell Counting Kit 8 (Gibco) assay to assess cell proliferation. We placed 2000 transfected cells per well into 96-well plates in triplicate wells for 5 days and detected cell proliferation at 1, 2, 3, 4, and 5 d after seeding according to the standard manufacturer’s instructions.
We used the Annexin V-APC/7-AAD Apoptosis Kit (MultiSciences; Hangzhou China) to detect apoptosis according to the manufacturer's instructions. In short, after washing in PBS, cells were incubated in 7-AAD staining solutions and APC Annexin-V in the dark at room temperature for 5 min. After incubation, the cells were collected by a C6 flow cytometer (BD, NY, USA) and FlowJo 10.4 software was used for analysis. Apoptotic cells population include 7-AAD- and APC Annexin-V +  (undergoing apoptosis) cells and 7-AAD + and APC Annexin-V +  (end of apoptosis or death) cells.

WT and mycn mutant embryos with ET33J1: EGFP transgenic backgrounds were collected at 3 dpf. For proliferation analysis, the embryos were immersed in 3 mg/mL BrdU in 0.3× Danieau buffer at 60 hpf overnight in the dark, then washed 3 times with PBST and digested with cold 1% trypsin to a single-cell suspension. The cells were incubated and labeled with BrdU primary antibody and Alexa Fluor 594–labeled secondary antibody. For apoptosis analysis, the cells were stained with the Annexin V-APC/7-AAD apoptosis kit (Multisciences) per the manufacturer’s instructions, and the signal was detected by a DxFLEX flow cytometer.

Stable cells were cultured in 6-well plates at a density of 1 × 10⁵ cells per well. After 48 h of culturing, the cells were harvested with or without a supernatant and stained using an Annexin V-APC/7-AAD apoptosis kit (MultiSciences, Hangzhou, China) and a cell cycle staining kit (MultiSciences, Hangzhou, China) according to the manufacturers’ instructions. Data were obtained using flow cytometry (Beckman Coulter, Brea, CA, USA) and analyzed with FlowJo v10.0 (BD Biosciences, Ashland, OR, USA) or CytExpert v2.4 (Beckman Coulter, Brea, CA, USA).

Cell apoptosis was assessed using the Annexin V-APC/7-AAD apoptosis kit (MultiSciences, China) according to the manufacturer’s instructions. Briefly, 3 × 10⁵ cells per well were seeded in 6-well plates and incubated in a 5% CO₂ atmosphere at 37 °C for 24 h. Imatinib (3 µM) or solvent was added to the culture medium for 48 h, after which the cells were collected. After being washed twice in PBS at 4 °C, cells were resuspended in binding buffer (500 µl). Annexin V-APC (5 µl) and 7-AAD (10 µl) were added to the suspension and the mixture was incubated for 5 min at 4 °C. The apoptosis index was examined by flow cytometry (ACCURI C6; BD, USA).

Apoptosis was evaluated using an Annexin V‐APC 7AAD apoptosis kit (MultiSciences Biotech). Flow cytometer (BD Biosciences) was used to quantify apoptosis detection and data analysis. Q1 represent dead cells, Q2 represent early apoptotic cells, Q3 represent viable cells, and Q4 represent late apoptotic cells.