Apc annexin 5 apoptosis detection kit with 7 aad

APC Annexin V Apoptosis Detection Kit with 7-AAD (BD Biosciences, San Jose, CA, USA) was used for the identification of apoptotic and necrotic cells. All experiments were taken according to manufacturer’s protocol. A549 and CORL-105 cells were collected, washed twice with cold PBS and then resuspend in 1× Binding Buffer at a concentration of 1 × 10⁶ cells/mL. Subsequently, 1 × 10⁵ cells were stained with addition of 5 µL of APC Annexin V and 5 µL of 7-AAD. After incubation (15 min. at 25 °C, in the dark), 400 µL of 1× Binding Buffer was added to each tube and analyzed by flow cytometry within an hour (Flow Sight^®, Amnis). In parallel, a set of control samples were used as follows:

unstained cells (wild type and after transduction with lentivectors with GFP marker),

apoptosis control: cells stained with APC Annexin V (cells that undergo apoptosis after addition of camptothecin, final concentration 6 µM, to 1 × 10⁶ A549 and CORL-105 wild type cells and incubation for 24 h at 37 °C),

necrosis control: cells stained with 7-AAD (A549, CORL-105 wild type cells incubated at 96 °C for 10 min.)

Experiments were prepared in triplicates. Analysis of flow cytometry results was performed using IDEAS^® (Image Data Exploration and Analysis Software, Amnis).

For the cell apoptosis assay, HCT116, DLD1, and RKO cells were stimulated with DHA (10 and 20 μM) for 48 h and then harvested for flow cytometric analysis (Beckman Coulter Cytoflex, Beckman, USA) using an APC Annexin V Apoptosis Detection Kit with 7-AAD (Cat No. 640930, BD Biosciences, USA) according to the manufacturer’s instructions.
For the cell cycle analysis, HCT116, DLD1, and RKO cells were treated with different DHA concentrations for 48 h and detached with trypsin after washing with cold PBS. Then, 70% ice-cold ethanol was used for cell fixation at 4°C overnight. After that, the cells were used for cell cycle analysis by a DNA Content Quantification Assay (Cell Cycle, Solarbio, China). Briefly, the cells were incubated with 100 μl of RNase at 37°C for 30 min, stained with 400 μl of propidium iodide (PI) for 30 min at 4°C in the dark, and then tested by flow cytometry. The data were analyzed by FlowJo 10.4 software (Becton, Dickinson & Company, NJ, USA).