Apoptosis detection kit

Female 4-week-old female nude mice were obtained from Slaccas Company. 2 × 10⁶ cells of each group, at 12 hours after transfection, were injected subcutaneously into the mice. For the same cell line, the normal control group was implanted into the left posterior flank and the knockdown group was in the right of the same mouse. The size of tumors were measured every week by caliper. 3 weeks later, the mice were executed and tumors were statistically analyzed. The apoptotic change of tumors were detected by the TUNEL staining using apoptosis detection kit (Vazyme Biotech, Nanjing, China). The tumors were made into paraffin sections and then settled according to the protocol. The samples were incubated with the TUNEL reagent containing BrightGreen Labeling Mix and Recombinant TdT Enzyme at 37°C for 1 hour. The samples were washed in 1X PBS and incubated with DAPI for 5 minutes. Fluorescence microscope at 10X magnification. The total number of both DAPI and TUNEL positive cells were counted from at least five random visual fields from each sample. Each experiment was repeated 3 times.

For the apoptosis assay, an apoptosis detection kit (Vazyme Biotech, Jiangsu, China) was used according to the manufacturer's instructions. Briefly, mouse bone marrow neutrophils were treated as indicated for 4 h, collected, washed twice, and resuspended in 1 × binding buffer at a concentration of 1 × 10⁶ cells/ml. Then, 5 μl Annexin V and 5 μl PI were added to the cell suspension, and the samples were incubated for 15 min in the dark. Apoptosis was determined by flow cytometry, and apoptotic cells were Annexin V-positive and PI-negative/positive.

Apoptosis was measured using an apoptosis detection kit (Vazyme, Nanjing, China). Cells were transfected for 48 h in 6‐well plates, stained with annexin V and propidium iodide, and analyzed using flow cytometry (BD FACSCalibur, Marshall Scientific, Hampton, NH). Data was analyzed using Cellquest software.