Cells were seeded in 24-well plates (2×105 per well) and cultured with medium containing 400 µM CoCl2 for 6 h. Apoptotic cells were measured by flow cytometry using Annexin V-APC and 7-AAD Apoptosis Detection reagent (BD Biosciences). Briefly, cell were digested, collected, washed and incubated at room temperature for 10 min with 1X binding buffer containing Annexin V-conjugated APC and 7-AAD. Annexin V-APC staining was used to detect exposure of phosphatidylserine. 7-AAD is able to penetrate the cells when cell apoptosis and cell death occurs. Annexin V-APC was used to detect early apoptotic cells, and Annexin V-APC and 7-AAD were used to detect necrotic cells or/and nonviable apoptotic cells. Cells were detected by BD Accuri C6 Flow Cytometer and analyzed by BD Accuri™ C6 Software version 1.0 (BD Biosciences).
Annexin 5 apc and 7 aad apoptosis detection reagent
Manufactured by BD
The Annexin V-APC and 7-AAD Apoptosis Detection reagent is a laboratory tool used to identify and quantify apoptotic cells. Annexin V binds to phosphatidylserine, a molecule that is exposed on the surface of cells undergoing apoptosis. 7-AAD is a fluorescent dye that can penetrate the cell membrane of late apoptotic or necrotic cells. The combined use of these two reagents allows for the detection and differentiation of viable, early apoptotic, and late apoptotic/necrotic cells.
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2 protocols using annexin 5 apc and 7 aad apoptosis detection reagent
1
Quantifying CoCl2-Induced Apoptosis by Flow Cytometry
2
Quantifying Cell Apoptosis by Flow
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Cell apoptosis was detected using the Annexin V-APC and 7-AAD Apoptosis Detection reagent (BD Biosciences) as previously described [21 (link)]. Briefly, 2 × 105 cells per well were plated into 6-well plates and treated with 800 µM CoCl2 for 48 h before treatment with nesfatin-1 at different concentrations (20, 60, and 80 nM) for another 24 h. Afterward, cells were washed and incubated Annexin V-conjugated APC and 7-AAD. Cell apoptosis was detected using a flow cytometer (Beckman, Pasadena, USA).
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