Pi apoptosis detection kit

Cell apoptosis was detected by fluorescein isothiocyanate (FITC)-labeled Annexin V/propidium iodide (PI) Apoptosis Detection Kit (556547, BD Biosciences, USA) according to the manufacturer’s instructions. Briefly, stably infected KGN cells were cultured in the medium supplemented with either 20 µM Pifithrin-α (PFT-α, Santa Cruz Biotech), 10 mM N-acetyl-l-cysteine, or neither. They were harvested after 6 h incubation, washed with ice-cold PBS, resuspended in 500 μl 1 × binding buffer supplemented with 5 μl Annexin-V-FITC and 1 μl PI for 30 min, and left in the dark at RT. Flow cytometric analysis was performed on a BD FACS Aria Fusion (BD Biosciences). Data acquisition and analysis were performed using FlowJo V8 software (TreeStar, USA).

The apoptotic cells were also evaluated using annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) Apoptosis Detection Kit (BD, USA), to examine early and late apoptotic changes as previously established. The samples were washed twice and adjusted to a 5 × 10⁵ cells/mL concentration with phosphate buffer saline (PBS). 200 μL of suspension was added into each labeled tube. 5 μL of annexin V-FITC and 10 μL PI (20 μg/mL) were then added into the labeled tube and incubated for at least 15 min at room temperature in the dark. 200 μL of cold PBS binding buffer was then added into each tube without washing and analyzed using the FCM analysis (BD, USA), as soon as possible (within 30 min). The apoptotic cells were defined as the population that was PI negative (indicating an intact plasma membrane) and Annexin V-FITC positive. This FCM assay was also done in triplicate.

HaCaT human keratinocyte cells were cultured in the presence of samples for 3 h, as previously described. Apoptosis-mediated cell death was examined using a double staining method with a fluorescein isothiocyanate (FITC)-labeled Annexin V/propidium iodide (PI) Apoptosis Detection kit (BD Pharmingen™, BD Biosciences, USA) according to the manufacturer’s instructions. Flow cytometric analysis was performed immediately after staining. Cells in the early stages of apoptosis were Annexin-V-FITC-positive, whereas cells that were both Annexin V-FITC and PI-positive were in the late stages of apoptosis.

Apoptosis rate of HAECs induced by TMAO was detected with Annexin V-FITC/propidium iodide (PI) Apoptosis Detection Kit (BD Biosciences). According to manufacturer’s instructions, the cells were harvested by centrifugation (300 g/5 min), washed with cold PBS and 1× binding buffer. After that, cells were resuspended in 200 μl 1 × binding buffer and incubated with Annexin V-FITC (5 μl) and PI (10 μl) at room temperature in the dark for 20 min. The cells were added 300 μl 1 × binding buffer and tested by flow cytometry (Cytoflex, Beckman Coulter, United States). The apoptosis ratio is equal to the sum of early and late apoptosis ratio.