Cell apoptotic rates were assessed by Annexin V-FITC/PI double staining assay (BD Biosciences, 556547) based on the manufacturer’s protocol. Cells were stained with 10 μL of PI dye and 5 μL of Annexin V dye for 15 min at room temperature. Apoptosis was analyzed using the BD AccuriTM C6 Plus flow cytometry. Cells in the second and third quadrants were considered apoptotic.
Annexin 5 fitc pi double staining assay
Manufactured by BD
Sourced in United States
The Annexin V-FITC/PI double staining assay is a laboratory technique used for the detection and quantification of apoptosis, a type of programmed cell death. The assay utilizes two fluorescent dyes: Annexin V-FITC and Propidium Iodide (PI). Annexin V-FITC binds to phosphatidylserine, which is exposed on the surface of apoptotic cells, while PI stains the DNA of cells with compromised cell membranes, indicating late-stage apoptosis or necrosis. This combined staining allows for the differentiation between viable, early apoptotic, late apoptotic, and necrotic cells.
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4 protocols using annexin 5 fitc pi double staining assay
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Annexin V-FITC Apoptosis Assay
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Assessing Cell Proliferation and Apoptosis
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The cell proliferation assay was performed as described before 24 (link). Briefly, CRC cells were transfected as indicated. After 12 h, 1 × 104 cells were seeded into 96-well plates, and medium containing oxaliplatin was added to each well. After 48h incubation, a CCK8 (Dojindo, Japan) assay was performed. The IC50 and the cell viability rate were calculated. Apoptosis analysis was performed using an Annexin V FITC/PI double staining assay (BD Biosciences, San Jose, CA) following the manufacturer's protocol.
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Apoptosis Detection in Cancer Cells
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An annexin V-FITC /PI double staining assay (BD, USA) was used to detect the apoptosis of A549 and XWLC-05 cells 12, 24, 48, 96, and 192 hr after 4 Gy and 8Gy (respectively) 6MV X-ray irradiation. The cells were washed twice with cold PBS and then resuspended in Annexin V Binding Buffer at a concentration of 1 × 106 cells/mL. Then, 100 μL of the solution (1 × 105 cells) was transferred to a 5 mL culture tube.
Next, 5 μL of FITC-annexin V and 5 μL PI were added with gentle vortexing and incubation for 15 min at room temperature (25°C) in the dark. Then, 400 μL of Binding Buffer was added to each tube and analyzed by flow cytometry within 1 hr, so as in tumor suspensions.
Next, 5 μL of FITC-annexin V and 5 μL PI were added with gentle vortexing and incubation for 15 min at room temperature (25°C) in the dark. Then, 400 μL of Binding Buffer was added to each tube and analyzed by flow cytometry within 1 hr, so as in tumor suspensions.
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Annexin V-FITC/PI Apoptosis Assay
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The induction of apoptosis was examined using the Annexin V-FITC/PI double staining assay (BD Biosciences; Becton-Dickinson and Company). Briefly, H9c2 cells were gently washed with ice-cold PBS, digested with 0.25% Trypsin without ethylenediaminetetraacetic acid (Gibco; Thermo Fisher Scientific, Inc.) and resuspended in complete culture medium. The cells were then centrifuged at 120 × g for 5 min at 4°C and the cell pellet was resuspended in ice-cold PBS and centrifuged at the same conditions. Finally, the cells were resuspended in 500 µl 1X binding buffer supplemented with 5 µl Annexin V-FITC and 5 µl PI. The cells were incubated at room temperature for 15 min in the dark and were analyzed by flow cytometry (BD FACSCalibur™; BD Biosciences; Becton-Dickinson and Company). Data was analyzed using FlowJo version 7.6.1 software (FlowJo LLC).
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