Annexin V/propidium iodide double staining (BD Biosciences, CA, USA) was used to detect cell apoptosis. HESFs were plated in 60-mm dishes (4 mL, 1 × 106/well) and incubated for 24 hours. After treatment with different concentrations of MMC, the detached and adherent cells were collected at the indicated time points and washed twice with ice-cold PBS. The cells were then resuspended in binding buffer at a concentration of 1 × 106/mL and incubated with annexin V-FITC and propidium iodide for double staining, according to the manufacturer's instructions. The mixture was incubated in the dark for 15 minutes at room temperature and analyzed using the Beckman Coulter FC500 flow cytometry system and CXP software (Beckman Coulter, Fullerton, CA, USA). The apoptosis rate in this study represents the total apoptosis rate, including early apoptosis rate and late apoptosis rate.
Annexin 5 propidium iodide double staining
Manufactured by BD
Sourced in United States
Annexin V/propidium iodide double staining is a laboratory technique used for the detection and quantification of apoptosis (programmed cell death) in cell populations. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, which is exposed on the outer cell membrane during the early stages of apoptosis. Propidium iodide is a fluorescent dye that binds to DNA and can only enter cells with compromised cell membranes, which occurs in the later stages of apoptosis or necrosis. The combined use of these two markers allows for the identification and quantification of cells in different stages of the apoptotic process.
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2 protocols using annexin 5 propidium iodide double staining
1
Annexin V/Propidium Iodide Apoptosis Assay
2
Apoptosis Evaluation via Annexin V/PI Staining
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Apoptosis was assessed using Annexin V/propidium iodide double staining according to the manufacturer (BD Biosciences, Franklin Lakes, NJ, USA). After treatments, cells were trypsinized and stained with 0.5 mg/mL Annexin V in binding buffer (10 mM HEPES free acid, 0.14 M NaCl, and 2.5 mM CaCl2) for 30 min. Afterward, propidium iodide (5 mg/mL final concentration) was added and incubated for another 15 min. Cells were then analyzed using a flow cytometer and BD FACSDiva software version 7 (both BD Biosciences).
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