Fitc annexin 5 apoptosis detection kit

Sperm plasma membrane phosphatidylserine externalization (the translocation of phosphatidylserine from the inner to the outer leaflet of the sperm plasma membrane) was detected with an Annexin-V-FITC apoptosis detection kit (TransGen, FA101, Beijing, China) as previously described [22 (link)]. After gibberellin exposure, sperm were collected, washed with cold PBS, centrifuged at 1,500 rpm for 3 min and resuspended in 1× Annexin V binding buffer (100 μL). Then, Annexin V (5 μL) and propidium iodide (PI) (5 μL) were added, and the samples were incubated for 15 min in the dark at room temperature. Lastly, 400 μL of binding buffer was added. Samples were analyzed on a FACSCalibur flow cytometer (BD Bioscience) within 30 min. Apoptotic cells were counted and reported as a percentage of the total cell count. The data were expressed as the mean ± SEM.

Bladder cancer T24 and 5637 cells were transiently transfected with siRNAs or plasmid vectors. 48 hours after transfection, cells were harvested and then collected. The FITC Annexin V Apoptosis Detection Kit (TransGen, Perking, China) was utilized to double stain cells with FITC-Annexin V and PI according to the manufacturer's instructions. Flow cytometry (EPICS, XL-4, Beckman, CA, USA) was utilized to observe cell apoptosis. In the graphs, cells were discriminated into dead cells, living cells, early apoptotic cells and late apoptotic cells. The ratio of early apoptotic cells was regarded as an observation index to compare the experimental group and negative group. Each experiment was done at least three times.

G361 and A375 cells were seeded in 6-well plates. 24H after transfection, the transfected cells were harvested using trypsin without EDTA. According to the manufacturer's protocols, the FITC Annexin V Apoptosis Detection Kit (TransGen, Beijing, China) could double stain cells with FITC-Annexin and PI. The ratio of early apoptotic cells in melanoma cells was determined by the flow cytometry (EPICS, XL-4, Beckman, CA, USA). The experiments were done at least three times.

Cell apoptosis was detected by Hoechst 33258 staining assay, caspase-3 enzyme linked immunosorbent assay (ELISA) and flow cytometry assay. For Hoechst 33258 staining assay, the Hoechst 33258 staining kit (Beyotime, Shanghai, China) was used to observe the apoptotic cells and measure the apoptosis ratio of cancer cells at 48h post-transfection of si-ZEB1-AS1 or si-NC. For ELISA assay, a Caspase-3 Colorimetric Assay kit (Abcam, Cambridge, UK) was used to measure the relative activity of cleaved caspase-3 in cancer cells at 48h post-transfection. For flow cytometry assay, bladder cancer cells were collected at 48h post-transfection and double stain the cancer cells with FITC-Annexin V and PI by utilizing FITC Annexin V Apoptosis Detection Kit (TransGen, Perking, China) following the manufacturer's instructions. After double staining, a flow cytometer (EPICS, XL-4, Beckman, CA, USA) was used to measure the apoptosis of bladder cancer cells. In the graphs, cells were distinguished into four regions which represented dead cells, living cells, early apoptotic cells and late apoptotic cells, respectively. The percentage of region which represented early apoptotic cells in the graph was measured and subjected to quantitative comparison. Experiments were repeated five times.

G361 and A375 cells were seeded in six-well plates. Then transfected cells were collected after incubation under light/dark for 24 h and harvested using trypsin without EDTA. According to the manufacturer’s protocols, the FITC Annexin V Apoptosis Detection Kit (TransGen, Beijing, China) could double stain cells with FITC Annexin and PI. The ratio of early apoptotic cells in melanoma cells was determined by the flow cytometry (EPICS, XL-4, Beckman, CA, United States). The experiments were done at least three times.