The fraction of apoptotic and necrotic cells was determined using annexin V and propidium iodine (A/PI) staining of cells and measured by flow cytometry. For analysis, trypsinized cells including the supernatant were collected, transferred into 15 mL falcon tubes, washed with PBS and stored on ice. For labeling, cells were incubated for 15 min at RT in 50 µL 1× annexin binding buffer containing 2.5 µL annexin V/FITC (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) on ice. For PI staining, 10 µL PI from a 1 mg/mL stock solution (Sigma-Aldrich, Steinheim, Germany) and annexin binding buffer were added to each sample. Cells were incubated for additional 10 min. Cells were kept in the dark until measurement. Data acquisition was performed using the FACS Canto II flow cytometer (Becton Dickinson GmbH, Heidelberg, Germany) and the data was analyzed using the BD FACSDiva software. Apoptotic cells were defined as annexin V+/PI− cells, whereas necrotic cells were defined as annexin V+/PI+ cells (see Figure S2 ). Experiments were performed in duplicate and repeated at least twice.
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2 protocols using 1 mg ml stock solution
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Quantifying Apoptosis and Necrosis
2
Quantifying Apoptosis and Necrosis by Flow Cytometry
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The fraction of apoptotic and necrotic cells was determined using annexin V and propidium iodide (A/PI) staining of cells and measured by flow cytometry. For analysis, trypsinized cells including the supernatant were collected, transferred into 15 mL falcon tubes, washed with PBS, and stored on ice. For labeling, cells were incubated for 15 minutes at RT in 50 μL 1× annexin binding buffer containing 2.5 μL annexin V/FITC (MiltenyiBiotec GmbH). For PI staining, 10 μL PI from a 1 mg/mL stock solution (Sigma-Aldrich) and annexin binding buffer were added to each sample. Cells were incubated for additional 10 minutes on ice. Cells were kept in the dark until measurement. Data acquisition was performed using the FACS Canto II flow cytometer (Becton Dickinson GmbH) and the data were analyzed using the Flowing Software 2 program (PerttuTerho, Turku Center for Biotechnology, University of Turku, Finland). Apoptotic cells were defined as Annexin V+/ PI− cells, whereas late apoptotic/necrotic cells were defined as Annexin V+/ PI+ cells. For the sake of simplicity, this fraction is simply called “necrosis.” A representative plot showing the Annexin V+/ PI− and Annexin V−/ PI+ fractions is shown in Supplementary Fig. S1.
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