The percentages of viable as well as apoptotic cells were quantified by flow cytometry using Annexin V-fluorescein isothiocyanate and propidium iodide (PI) staining (BD Biosciences, San Diego, CA). Cells were stained with Annexin-V/FITC and PI in a binding buffer along with the appropriate control. Total number of Annexin V and PI positive cells were counted using a FACScan flow cytometer (BD Biosciences). The data was analyzed using software (FlowJo version 10; FloJo Data Analysis Software, Ashland, OR).
Annexin 5 fluorescein isothiocyanate and propidium iodide staining
Manufactured by BD
Annexin V-fluorescein isothiocyanate and propidium iodide (PI) staining is a laboratory technique used to detect and quantify apoptosis, or 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 membrane of apoptotic cells. Fluorescein isothiocyanate (FITC) is a fluorescent dye that is conjugated to Annexin V, allowing for the detection of apoptotic cells by flow cytometry or fluorescence microscopy. Propidium iodide (PI) is a DNA-binding dye that is used to detect necrotic or late-stage apoptotic cells, as it can only enter cells with compromised membranes.
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4 protocols using annexin 5 fluorescein isothiocyanate and propidium iodide staining
1
Apoptosis Quantification by Flow Cytometry
2
Apoptosis Quantification by Flow Cytometry
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The percentages of viable as well as apoptotic cells were quantified by flow cytometry using Annexin V-fluorescein isothiocyanate and propidium iodide (PI) staining (BD Biosciences, San Diego, CA). Cells were stained with Annexin-V/FITC and PI in a binding buffer along with the appropriate control. Total number of Annexin V and PI positive cells were counted using a FACScan flow cytometer (BD Biosciences). The data was analyzed using software (FlowJo version 10; FloJo Data Analysis Software, Ashland, OR).
3
Apoptosis Detection in Mouse Kidney
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Apoptosis was tested with TUNEL (Roche) in mouse kidney sections and through annexin V–fluorescein isothiocyanate and propidium iodide staining (BD Biosciences) in cultured TECs.
4
Apoptosis Evaluation in Glioma Stem Cells
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After CoCl2 application or 14-3-3β siRNA transfection, GSC cell apoptosis rate was evaluated by annexin V-fluorescein isothiocyanate and propidium iodide staining (BD Biosciences) for 15 min at RT (25°C) in the dark and flow cytometry analysis (BD, FACSuite Research Assay Software). Bax/Bcl-2 assessment is a way of assessing cell apoptosis [46 (link), 47 (link)]. Thus, Bax/Bcl-2 protein expression and mRNA levels were also detected by western blotting or qPCR following 14-3-3β siRNA transfection combined with CoCl2 treatment (anti-Bax and anti-Bcl-2, 1 : 1,000; Santa Cruz Biotechnology, Inc.). In addition, caspase-3 activity was measured to determine GSC cell apoptosis after above treatments by using a kit (Promega, Inc.) according to the manufacturer's instructions [48 (link)].
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