Apoptotic cells were quantified using an Annexin V-FITC-propidium iodide (PI) double staining kit (Cat. No. 556547 BD Pharmingen, San Jose, CA, USA) according to the manufacturer’s instructions. Each of the HCT-116 and Caco-2 cells was plated in a 24-well plate (5 ´ 105 cells/well) and then incubated for 24 hours for attachment, and then the cells were treated with Q and/or 5-FU, as in the experimental design, for 48 hours. After cell collection by trypsinization, HCT-116 and Caco-2 cells were washed with PBS and stained by annexin V/propidium (BD Biosciences) based on the manufacturer’s instructions for 25 minutes at room temperature in a dark place. The stained cells were analysed using an Attune flow cytometer (Applied Bio-system, USA). Experiments were performed in triplicate.
Annexin 5 propidium
Manufactured by BD
Sourced in United States
Annexin V/propidium is a laboratory reagent used for the detection and analysis of apoptosis (programmed cell death) in cell samples. It is a combination of two dyes: Annexin V, which binds to phosphatidylserine exposed on the surface of apoptotic cells, and propidium iodide, which stains the DNA of cells with compromised cell membranes. This reagent allows for the identification and differentiation of viable, early apoptotic, and late apoptotic/necrotic cells through flow cytometry or fluorescence microscopy.
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Lab products found in correlation
2 protocols using annexin 5 propidium
1
Quantifying Apoptosis in Cancer Cells
2
Apoptosis Induction in PC3 Cells
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PC3 cells (2×105 per well) were seeded into a 6-well plate in RPMI 1640 medium supplement with 10% FBS, 1% pen/strep and incubated overnight in 5% CO2 incubator at 37°C and 95% humidity. Then, the cells were treated with 5-FU (0.75 μM) and rutin (700 μM) or combination of 5-FU and rutin (0.75 μM and 700 μM respectively) for 48 hours. Subsequently, the cells were collected by trypsinization and washed with PBS and stained by Annexin V/propidium (BD Biosciences) based on the manufacturer’s instructions for 25 minutes at room temperature in a dark place.25 (link) The stained PC3 cells were analyzed using a flow cytometer (FACScan; Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). Experiments were performed in triplicates.
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PC3 cells (2×105 per well) were seeded into a 6-well plate in RPMI 1640 medium supplement with 10% FBS, 1% pen/strep and incubated overnight in 5% CO2 incubator at 37°C and 95% humidity. Then, the cells were treated with 5-FU (0.75 μM) and rutin (700 μM) or combination of 5-FU and rutin (0.75 μM and 700 μM respectively) for 48 hours. Subsequently, the cells were collected by trypsinization and washed with PBS and stained by Annexin V/propidium (BD Biosciences) based on the manufacturer’s instructions for 25 minutes at room temperature in a dark place.25 (link) The stained PC3 cells were analyzed using a flow cytometer (FACScan; Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). Experiments were performed in triplicates.
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