Apoptotic and dead cell counts were performed using FITC-labeled Annexin V and PI staining (BD Biosciences, San Jose, CA, USA) by flow cytometry. The cells were collected and resuspended in binding buffer at a concentration of 3×106/mL; then, 100 µL of cell suspension was added to 5 µL of Annexin V–FITC and 10 µL of PI and mixed for 15 minutes in dark at room temperature. Next, 400 µL of phosphate-buffered saline was added to the solution. A FACScan instrument (BD Biosciences) was used to count the cells (1×103) at an excitation wavelength of 490 nm. The CellQuest software (BD Biosciences) was used for data collection and processing.
Fitc labeled annexin 5 and pi staining
Manufactured by BD
Sourced in United States
FITC-labeled annexin V and PI staining is a laboratory tool used for the detection and analysis of apoptotic cells. It utilizes the binding properties of annexin V, a protein that binds to phosphatidylserine exposed on the surface of apoptotic cells, and propidium iodide (PI), a dye that stains the nucleic acids of cells with compromised cell membranes. This combination of stains allows for the identification and quantification of different cell populations, including viable, early apoptotic, and late apoptotic/necrotic cells.
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4 protocols using fitc labeled annexin 5 and pi staining
1
Apoptosis and Dead Cell Quantification
2
Apoptosis and Cell Viability Analysis
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For clonogenic survival assays, cells plated into petri dishes (60 mm × 15 mm) (2,500 cells per 10-cm plate) and grown for 7–10 days, fixed, and stained by crystal violet. The number of colonies (>50 cells) was counted. For the MTT assay, cells were plated into 96-well plates. Cell growth was monitored 96 h after transfection by the MTT [(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium] (Sigma) assay. Apoptotic and dead cell counts were performed using FITC-labeled annexin V and PI staining (BD Pharmingen, San Diego, CA, USA), followed by flow cytometry (Becton Dickinson, San Jose, CA, USA). Briefly, the cells were gently vortexed and resuspended in binding buffer at a concentration of 3 × 106/ml, and then 100 μl of cell suspension was added to 5 μl of annexin V-FITC and 10 μl of PI and mixed for 15 min in the dark at room temperature. Next, 400 μl of PBS was added to the solution. A FACScan instrument (BD Biosciences) was used to count the cells (1 × 103) at an excitation wavelength of 490 nm. BD FACSDiva 4.0 Software was used for data collection and processing.
3
Apoptosis and Cell Death Quantification
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Apoptotic and dead cell counts were performed using FITC-labeled annexin V and PI staining (BD Biosciences), followed by flow cytometry. The cells were gently vortexed and resuspended in binding buffer at a concentration of 3 x106/ml, and then 100 μl of cell suspension was added to 5 μl of annexin V-FITC and 10 μl of PI and mixed for 15 min in the dark at room temperature. Next, 400 μl of PBS was added to the solution. A FACScan instrument (BD Biosciences) was used to count the cells (1x103) at an excitation wavelength of 490 nm. CellQuest software was used for data collection and processing.
4
Apoptosis Detection via Flow Cytometry
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Flow cytometry was performed with FITC-labeled annexin V and PI staining (BD Pharmingen) to detect phosphatidylserine externalization as an endpoint indicator of apoptosis according to the manufacturer's instructions.
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