Annexin 5 fitc

Apoptosis and the cell cycle were assayed via flow cytometry (Beckman CytoFLEX, Brea, CA, USA). A total of 4 × 10⁵ cells were washed twice with PBS before being resuspended in 100 μL of 1× binding buffer and 5 μL of Annexin V-FITC (Transgen biotech, Guangzhou, China). Then, the samples were incubated with 5 μL of propidium iodide (PI) for 15 min before apoptosis was measured. For the cell cycle, 4 × 10⁵ cells were washed twice with phosphate buffer saline (PBS) and then fixed with chilled 75% ethanol overnight. Then, the cells were incubated with 50 μL of RNase and 300 μL of PI for 30 min before flow cytometry. The CytExpert 2.0 (Beckman, Brea, CA, USA) and ModFit LT5.0 (Verity Software House, Topsham, ME, USA) were used to analyze the apoptosis and the cell cycle, respectively.

5637 and EJ cells were seeded into 6-well plates. After being treated with MTX-211 for 48 h, cells were enzymatically dissociated and washed three times with cold PBS. To evaluate apoptosis events, the cells were stained with a combination of 5 μL PI (20 μg/mL) and 5 μL Annexin V/FITC (50 μg/mL) (FA101-01, Transgen, Beijing, China) in PBS for 15 min at room temperature in the dark [51 (link)]. On the other hand, for the analysis of cell cycle, cells were fixed in 75% alcohol overnight at 4 °C. Then, after incubating with 20 μL RNase A for 30 min at 37 °C, cells were stained with 400 μL PI (100 μg/mL) (BB-4104, Bestbio, Shanghai, China) in PBS for 30 min in the dark at 37 °C the next day. Subsequently, both sets of stained cells were analyzed using flow cytometry, and the results were processed and analyzed using Flowjo software (v 10.4).

Cells were irradiated with 4 Gy γ-rays, and after 48 h, the cells were washed with cold PBS triply and then collected in 50 µL of suspension buffer containing 2.5 µL Annexin V-FITC and 2.5 µL PI (TransGen Biotech, Beijing, China). Then, 200 µL buffer was added to the solution after incubation for about 15 min in the dark at room temperature; then, cell apoptosis was detected using flow cytometry (Beckman CytoFLEX, CA, USA).

After 24 h of irradiation with 4 Gy X-rays, the cells were washed with cold PBS and collected in 50 µL of the suspension buffer containing 2.5 µL Annexin V-FITC and 2.5 µL PI (TransGen Biotech, Beijing, China), then 200 µL buffer was added to the solution. After incubation for about 15 min in the dark at room temperature, cell apoptosis was detected using flow cytometry (Beckman CytoFLEX, CA, USA) and analyzed with FlowJo software (version 10). Viable cells display negative FITC Annexin-V and PI (FITC^-/PI^-), cells in early apoptosis have positive FITC Annexin-V and negative PI (FITC⁺/PI^-), cells in late apoptosis have positive FITC Annexin-V and PI (FITC⁺/PI⁺), and dead cells have negative FITC Annexin-V and positive PI (FITC^-/PI⁺).

The spermatozoa plasma membrane phosphatidylserine (PS) externalization was detected by Annexin-V-FITC (Cat #: FA101; Beijing TransGen Biotech Co., Ltd.; Beijing, China) to specifically detect PS translocation from the inner to the outer leaflet of the boar spermatozoa plasma membrane as described by Hurtado de Llera et al.28 (link). Briefly, after 24 hr treatment, boar spermatozoa cells were collected and resuspended in 1× Annexin V binding buffer (100 μl). Then 5 μl Annexin V and 5 μl PI were added into the samples following by incubation for 15 min in the darkness at RT. After incubation, 400 μl of binding buffer were added to each sample and mixed before flow cytometry analysis. The fluorescence values of probes Annexin V-FITC and PI were collected in the FL1 and FL3 sensors using a 520 and 620 nm bad pass filter, respectively. The results are expressed as the average of the percentage of Annexin V⁺/PI⁻ spermatozoa ± SEM.

After treatment with ISL for 24 h, the cells were collected and washed twice with cold PBS. Next, 5 μL of Annexin V–FITC (TRANSGEN), 5 μL of propidium iodide (PI) (TRANSGEN) and 50 μL of 1× binding buffer were added to each tube. The solution was then gently mixed and incubated at room temperature for 15 min in the dark. Then, 200 μL of 1× binding buffer was added to each tube. In addition, the cells were treated with 75% ethanol at 4 °C for 12–24 h, followed by staining with PI (BD Biosciences, Franklin Lakes, NJ, USA) for the cell cycle detection. Finally, flow cytometry (ACEA NovoCyte, Santa Clara, CA, USA) was used to explore cell apoptosis and cycle distribution [59 (link)].