Annexin 5 fitc apoptosis detection kit

For cell apoptosis analysis, the present assay was executed with Annexin V-FITC Apoptosis Detection Kit (Beyotime) referring to the user’s manual. AGS/DDP and MKN-45/DDP cells were harvested after transfection. Following re-suspension into single cell, Annexin V-FITC and PI were pipetted to dye the cells in darkness. Subsequently, apoptotic cells (Annexin V +) were examined exploiting flow cytometer (BD Biosciences, San Jose, CA, U.S.A.).

HaCaT cells had been treated with ALA-PDT by the abovementioned process. Then, these cells were harvested and washed with PBS. Finally, these cells were double-stained with an Annexin-V-FITC apoptosis detection kit (Beyotime Biotechnology, Shanghai, China). The cells were incubated with PI (10 μL) and Annexin-V-FITC (5 μL) at room temperature for 15 min in the dark. Then, they were quantitatively analyzed with a FACScan flow cytometer (BD, Franklin Lakes, NJ, USA). Cells stained with Annexin-V(+)/PI(−) were apoptotic in nature.

Cell apoptosis was determined by using an annexin V-FITC apoptosis detection kit (catalog number C1062M; Beyotime). Briefly, 195 μL of an annexin V-FITC binding solution was added to resuspend the cells gently. Next, 5 μL of annexin V-FITC and 10 μL of a propidium iodide (PI) staining solution were added, followed by gentle mixing and incubation at room temperature in the dark for 10 to 20 min. Flow cytometry analysis was conducted within 1 h. Early apoptosis was defined as annexin V-FITC singly positive cells (third quadrant [Q3]), and late apoptosis was defined as annexin V-FITC and PI doubly positive cells (Q2); the percentage of apoptotic cells was measured as the sum of Q2 and Q3. This gating strategy was applied to all flow cytometry analyses in this study. The flow cytometry assays were performed using an LSRFortessa X-20 cell analyzer (BD Biosciences), and the data were analyzed using FlowJo v10.6.2.

Apoptosis was analyzed using the Annexin V-FITC Apoptosis Detection Kit (Beyotime, Nantong, China) according to the manufacturer's protocol. ARPE-19 cells were seeded in the 6-well plates at a density of 4 × 10⁶ cells/ml. At 48 h after drug treatment, cells were harvested, and analyzed using a flow cytometer (FACS Partec, Germany) according to the manufacturer's protocol.

For apoptosis assay, equal numbers of cells were grown in 6-well plates (0.4 × 10 (6 (link)) cells/well) and treated with OXL over 48 h. The adhering cells were collected at the end of treatment and washed with 2% FBS in PBS. We performed flow cytometry to determine apoptosis based on the manual of the Annexin V-FITC Apoptosis Detection Kit (Beyotime). The percentages of AnnexinV-FITC/PI-stained cells were analyzed by the Flowjo software.

Cell apoptosis and cell cycle were detected with the Annexin V-FITC Apoptosis Detection Kit (C1062S, Beyotime Biotechnology, Shanghai, China) and the Cell Cycle and Apoptosis Analysis Kit (C1052, Beyotime Biotechnology), performed according to the manufacturer’s instructions [17 ]. Cell apoptosis and cell cycle were measured and analyzed by a flow cytometry machine (FACS Calibur™, BD Biosciences, San Jose, CA, USA).

Cell apoptosis was measured using an Annexin V-FITC Apoptosis Detection Kit (Beyotime). Cells were harvested, washed with PBS solution, and resuspended with 195 μl binding buffer softly; 5 μl Annexin V-FITC and 10 μl PI were added and incubated in a dark room for 15 min. 30,000 cells of each group were measured by a LSRll flow cytometer and BD FACSDiva software version 9 (BD Bioscience) and analyzed with FlowJo software (FlowJo, LLC). Cells were dicscriminated from debris and clumps using the FSC-A/SCC-A gating strategy based on experience. Only single cells were used by using an FSC-H/FSC-A gating strategy and selecting cells along the diagonal. The gating strategy is provided in Supplementary Fig. 2e.