The cell surface exposure of phosphatidylserine and the plasma membrane impairment of cells were assessed using Annexin V-FITC/propidium iodide (PI) Apoptosis Detection Kit (BioLegend, San Diego, CA, USA). Briefly, suspension of treated/control ARPE-19 cells, containing 5 × 105 cells, was washed with PBS and re-suspended in 0.5 ml cold binding buffer. Then, 1 μl of Annexin V-FITC and 2 μl of propidium iodide (PI) were added and the cells were incubated in the dark humidified incubator at 37 °C and 5% CO2 for 30 min. Following incubation, the cells were centrifuged at 100×g for 5 min and the supernatant was removed. The cell pellet was re-suspended in 0.5 ml cold binding buffer, and 10 μl of the 30 μg/ml PI solution was added. Cell samples were placed on ice, away from light, and FITC and PI fluorescence were immediately measured by using flow cytometer (FACSCalibur, Franklin Lakes, NJ, USA). Data were analyzed using Cell Quest Pro software (Becton Dickinson, Franklin Lakes, NJ, USA). The populations of live cells, early apoptotic cells, late apoptotic and necrotic cells were determined.
Annexin 5 fitc propidium iodide apoptosis detection kit
Manufactured by BioLegend
Sourced in United States
The Annexin V-FITC/propidium iodide (PI) Apoptosis Detection Kit is a tool used to detect and quantify apoptosis, a form of programmed cell death. The kit contains Annexin V-FITC, a fluorescent dye that binds to phosphatidylserine, and propidium iodide, a dye that stains DNA. These dyes can be used in flow cytometry or fluorescence microscopy to identify and distinguish between viable, early apoptotic, and late apoptotic/necrotic cells.
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2 protocols using annexin 5 fitc propidium iodide apoptosis detection kit
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Annexin V-FITC/PI Apoptosis Assay
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Apoptosis Assessment via Annexin V-FITC/PI Assay
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Since such an induced apoptotic process takes time to go through the Annexin assay was conducted after 72 hrs of the transfection. HCT116 and SW480 cells were seeded (250,000 cells/well) in 24-well plates and the apoptosis was measured using the Annexin V-FITC/propidium-iodide (PI) Apoptosis Detection kit (BioLegend) according to the manufacturer's protocol. Following the transfection with wt and M51R M-protein plasmids and incubation for 72 hrs, the cells were washed twice with ice-cold PBS. 1X binding buffer, followed by 10 µL of Annexin-V-FITC, and Subsequently, 10 µL PI was added to each microtube and incubated for 30 min at 4°C in a dark chamber. The apoptosis was analyzed using ow cytometry (BD accuri c6). In the ow-cytometry analysis of Annexin V-FITC/PI double staining, the late apoptotic or necrotic cells were visible in the upper right (UR) and early apoptotic cells in the lower right (LR) quadrants, and live cells in the lower left (LL) quadrants.
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