Pharmingen fitc annexin 5 apoptosis detection kit

The mode of cell death induced by MEDL was evaluated using the BD Pharmingen Annexin V-FITC Apoptosis Detection Kit (San Diego, CA, USA) following the manufacturer’s protocol. Cells at a density of 5 × 10⁵ cells in 4 mL medium were seeded in a 25 cm³ flask. The cells were then treated with IC₅₀ values of MEDL and 5-FU for 24 and 48 h. After the incubation period, all adhering and floating cells were harvested and transferred into sterile centrifuge tubes. The cells were centrifuged at 400g for 5 min at room temperature. The supernatant was aspirated, leaving approximately 50 µL of residual fluid in the tubes to avoid disturbing the pellets. FITC Annexin V (5 µL) and PI (5 µL) were added into the tubes containing cell pellets. The mixtures were gently vortexed and incubated for 15 min at room temperature in the dark. Binding buffer (400 µL) was added into each tube. The samples were analyzed using the BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA) within an hour.

To examine the apoptosis of human iPSCs induced by DNA damage, cells were seeded on a 6-well cell culture plate and pre-treated with 0.2 μM doxorubicin for 6 h. Cells were harvested and washed with ice-cold PBS and stained using the BD Pharmingen™ Annexin V-FITC Apoptosis Detection Kit (BD, Franklin Lakes, NJ, USA) according to the manufacturer’s protocol. Annexin V-FITC and propidium iodide (PI)-stained cells were detected using an LSRFortessa X-20 cell analyzer (BD Bioscience), and the data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA).

Pharmingen annexin V-FITC apoptosis detection kit (BD) was utilized to detect cell apoptosis. Briefly, BMDMs were treated with different concentrations of genipin for 24 h. Subsequently, cells were harvested and stained with PE-annexin V and 7-AAD for 15 min at room temperature. Flow cytometry (Agilent NovoCyte Advanteon BVYG) analysis was employed to measure cell apoptosis.

The number of apoptotic cells following treatment with plumbagin entrapped in transferrin‐bearing liposomes was determined using BD Pharmingen^® FITC Annexin V apoptosis detection kit (BD Biosciences), as described in the manufacturer's instructions. Cells were seeded in 6‐well plates at a density of 2 × 10⁵ cells per well and grown for 24 h before being treated with plumbagin (1 μg per well) entrapped in Tf‐bearing liposomes, control liposomes, or in solution. After 4 h treatment, the cells were harvested and centrifuged at 2000 rpm (370 g) for 5 min using an IEC Micromax^® centrifuge (ThermoFisher Scientific). Subsequently, the cell pellets were resuspended in 200 μL 1× Annexin V Binding Buffer (10× of the buffer containing 0.1‐M Hepes/NaOH (pH 7.4), 1.4‐M NaCl, and 25‐mM CaCl₂). Cell suspension (100 μL) was then transferred to a 5‐mL culture tube, followed by 5 μL of Annexin V‐FITC labelling reagent and 5 μL of propidium iodide and incubated for 15 min at 20°C protected from light. After incubation, 400 μL of Annexin V Binding Buffer was added to each tube before analysis of apoptosis using a FACSCanto^® flow cytometer (BD Biosciences). Ten thousand cells (gated events) were counted for each sample. The results were reported as percentages of specific cell populations (live cells, cells in early apoptosis, late apoptosis, and necrosis).

Cells were seeded into 96-well plates and treated as indicated. The CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay kit (Promega, Beijing, China) was used for cell viability analysis. Both western blotting and flow cytometry analysis were used for the detection of apoptosis. For flow cytometry analysis, the cells were collected by trypsinization and centrifugation at 1000 × g for 5 min and stained with the BD Pharmingen FITC Annexin V Apoptosis Detection Kit (BD Biosciences, Shanghai, China). The samples were analyzed with a BD FACSCalibur^TM flow cytometer (BD Biosciences) within 1 h.

Three different cell lines (SeFB, Ha-E, and Sf9) were seeded in 6-well plates at a density of 1 × 10⁶ cells per well in 1 mL of medium and allowed to attach for 1 h at RT. One milliliter of HvAV-3h- containing medium was added to each well to perform the ascovirus infection according to the procedures described above. The control cells or HvAV-3h-infected cells were incubated with 100 μg/mL H₂O₂, 5 μg/mL actinomycin D (ActD; Solarbio, Beijing, China), 5 μg/mL cMYC inhibitor (10058-F4; Beyotime, Shanghai, China), or 4 μg/mL TNF-α plus SM-164 apoptosis inducers (T/S, Beyotime, Shanghai, China) at 24 hpi. The treated cells were collected at 24 hpe. Cells treated with 5 μg/mL dimethyl sulfoxide (DMSO; Beyotime, Shanghai, China) were used as controls. The collected cells were washed with phosphate-buffered saline (PBS; 0.2 M, pH 7.4) three times, followed by staining with the BD Pharmingen FITC annexin V apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Fluorescence intensity tests were conducted using a flow cytometer (D×P Athena; Cytek, Fremont, CA, USA).