Cell apoptosis of transfected U87 or U251 cells with or without treatment were studied with a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) plus double Annexin V/PI staining (Invitrogen). Simply, transfected U87 or U251 cells with or without treatment were harvested from 6-well plates through centrifugation and mixed with 1X binding buffer (Invitrogen), after which were dyed by FITC-Annexin V and propidium iodide (PI). Apoptotic cells were assayed by flow cytometry.
Double annexin 5 pi staining
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The double Annexin V/PI staining is a laboratory technique used for the detection and quantification of apoptotic and necrotic cells. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, which is exposed on the cell surface during the early stages of apoptosis. Propidium iodide (PI) is a fluorescent dye that can only penetrate the cell membrane of cells with compromised integrity, such as necrotic or late-stage apoptotic cells. By using both Annexin V and PI, this staining method can distinguish between early apoptotic, late apoptotic, and necrotic cells.
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2 protocols using double annexin 5 pi staining
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Annexin V-PI Apoptosis Assay
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Apoptosis Detection by Annexin V/PI Staining
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The cell apoptosis were assessed by a flow cytometer (BD FACSCalibur; BD Biosciences) with double Annexin V/PI staining (Invitrogen). In brief, approximately 3 × 105 transfected cells were harvested from a 6-well plate by centrifugation and mixed with 500 μl of 1X binding buffer, followed by staining with 5 μl of FITC-Annexin V and propidium iodide (PI). The early apoptotic (Annexin V+/PI-) and late apoptotic (Annexin V+/PI+) cells were analyzed by flow cytometry and the total apoptotic rate was calculated in each group.
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