To determine cell apoptotic rates, the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining kit (BD Biosciences) was used according to the manufacturer's instructions. In brief, the cells were detached from the culture flasks with 0.05% trypsin and washed twice with chilled phosphate-buffered saline (PBS). The cells were then resuspended in 100 µl 1X binding buffer and stained with 5 µl Annexin V-FITC, which stains apoptotic cells (early apoptotic Q2 and late apoptotic Q3 quadrant), and PI for 15 min at room temperature in the dark. The samples were analyzed using a flow cytometer (BD FACSCalibur, BD Biosciences) and the data were analyzed using FlowJo 7.6 software.
Annexin 5 fluorescein isothiocyanate fitc propidium iodide double staining kit
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The Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining kit is a laboratory tool used to detect and quantify apoptosis and necrosis in cell samples. The kit contains Annexin V-FITC, which binds to phosphatidylserine exposed on the surface of apoptotic cells, and propidium iodide, which stains the nuclei of necrotic cells. This combination of fluorescent dyes allows for the identification and differentiation of live, apoptotic, and necrotic cells through flow cytometry analysis.
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5 protocols using annexin 5 fluorescein isothiocyanate fitc propidium iodide double staining kit
1
Annexin V-FITC/PI Apoptosis Assay
2
Apoptosis and Necrosis Assessment
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An annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double
staining kit (BD Biosciences, San Jose, CA, USA) was used to identify apoptotic
and necrotic cells. Both cell lines were exposed to different concentrations of
PL and VC, either alone or in combination, for 48 h. After incubation, cells
were collected and resuspended in 500 μl binding buffer and then stained with
5 μl of annexin V-FITC and 5 μl of PI in the dark for 15 min. The proportions of
apoptotic and necrotic cells were detected using an Accuri C6 flow cytometer (BD
Biosciences). Cells showing up as annexin V–/PI– were identified as healthy
cells, annexin V–/PI+ were identified as necrotic cells, annexin V+/PI– were
identified as early apoptotic cells and annexin V+/PI+ were identified as late
apoptotic cells.
staining kit (BD Biosciences, San Jose, CA, USA) was used to identify apoptotic
and necrotic cells. Both cell lines were exposed to different concentrations of
PL and VC, either alone or in combination, for 48 h. After incubation, cells
were collected and resuspended in 500 μl binding buffer and then stained with
5 μl of annexin V-FITC and 5 μl of PI in the dark for 15 min. The proportions of
apoptotic and necrotic cells were detected using an Accuri C6 flow cytometer (BD
Biosciences). Cells showing up as annexin V–/PI– were identified as healthy
cells, annexin V–/PI+ were identified as necrotic cells, annexin V+/PI– were
identified as early apoptotic cells and annexin V+/PI+ were identified as late
apoptotic cells.
3
Quantifying Cellular Apoptosis Using AnnexinV-FITC/PI Assay
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Cell apoptosis was determined using an AnnexinV-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-staining kit (BD Biosciences, USA), according to the manufacturer’s instructions. Ea.hy 926 cells were cultured in 6-well plates (1 × 106 cells/well) for 24 h. After treatment with LPS and melatonin, the cells were digested with ethylenediaminetetraacetic acid (EDTA)-free trypsin, stained with AnnexinV-FITC and PI, and subjected to flow cytometry using FACSCanto™ II (BD Biosciences). FlowJo V10 software (Tree Star, USA) was used for data analysis. Cells that were negative for both Annexin V-FITC and PI were considered viable. Annexin V-FITC positive and PI negative cells were recognized as early apoptosis; Annexin V-FITC and PI positive cells were late apoptosis; and Annexin V-FITC negative and PI positive cells were necrotic cells. Early and late apoptotic cells were used to calculate the percentage of apoptotic cells.
4
Apoptosis Quantification Using Annexin V-FITC/PI
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Apoptosis was examined using an Annexin V‐ fluorescein isothiocyanate (FITC)/propidium iodide (PI) double‐staining kit (BD Biosciences, USA), following the manufacturer's protocol. HCAECs were cultured into 6‐well plates (2 × 105 cells/well) for 48 h. After melatonin and TNF‐α treatment, the cells were digested with ethylenediaminetetraacetic acid (EDTA)‐free trypsin. Next, the cells were stained with FITC and PI, and subjected to flow cytometry using FACSCanto™ II (BD Biosciences). The data were analysed using FlowJo V10 software (Tree Star, USA).
5
Assessing Lung Cancer Cell Apoptosis
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The nuclear morphology of the A549 lung cancer cells was assessed by fluorescence microscopy following treatment of the cells to cell-permeable Hoechst 33342 dye. A total of 10 fields with 100 cells/field were selected randomly for measurement of the cells with condensed nuclei. Then, Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining kit (BD Biosciences, San Jose, CA, USA) was used for the determination of the percentage of the apoptotic lung cancer cells, as described previously (10 (link)) A flow cytometer, (BD Biosciences) and BD FACSuite software version 1.0 (BD Biosciences) for were used for analysis.
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