To assess the distribution of cells in different phases of the cell cycle, a flow cytometric analysis of the cell cycle distribution was performed. After transfection of the corresponding miRNA or dsRNA for 72 h, the cells were harvested and fixed with 70% cold ethanol overnight. Then, the cells were incubated with RNase A at 37°C for 30 min and resuspended in prop-idium iodide (PI; Cell Cycle Detection kit; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) at 4°C for 30 min. Then, the samples were analysed for PI fluorescence intensity by flow cytometry to assess the relative cellular DNA content, and the data were interpreted using CellQuest software (version 3.3; BD Biosciences). Apoptosis was measured by flow cytometry following staining of cells with Annexin V-fluorescein isothiocyanate (FITC) and PI in accordance with manufacturer's recommendations [Multisciences (Lianke) Biotech Co., Ltd., Hangzhou, China]. The cell apoptosis rate was calculated as follows: Cell apoptosis rate (%) = (apoptotic cell number ⁄ total cell number) ×100.
Annexin 5 fluorescein isothiocyanate fitc
Manufactured by MultiSciences Biotech
Sourced in China
Annexin V-fluorescein isothiocyanate (FITC) is a laboratory reagent used for the detection and quantification of apoptosis in cells. It binds to phosphatidylserine, which is exposed on the cell surface during the apoptotic process. The FITC fluorescent label allows for the visualization and analysis of apoptotic cells using flow cytometry or fluorescence microscopy.
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2 protocols using annexin 5 fluorescein isothiocyanate fitc
1
Cell Cycle Analysis and Apoptosis Quantification
2
Apoptosis Detection in HCC Cells
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After 48 h of transfection, the cells were harvested and washed with ice-cold phosphate-buffered saline (PBS). Next, the HCC cells were resuspended in 500 µL of 1× binding buffer, 10 µL of propidium iodide and 5 µL of Annexin V-fluorescein isothiocyanate (FITC; MultiSciences Biotech, Hangzhou, China) to detect cell apoptosis in a FACSCalibur flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Data analysis was performed using FCS express version 3 software (De Novo Software, Los Angeles, CA, USA). Experiments were performed three times.
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