HepG2 and SMMC-7721 cells were seeded at a density of 2 × 105/mL in six-well plates. For apoptosis rate analysis, after 24 h cells were exposed to Evo with different concentrations (0, 0.5, 1 µM) for another 24 h. Cells were then collected and washed twice with PBS to remove the medium. At least 1 × 105 cells were resuspended in 100 µL binding buffer containing Annexin V-FITC/propidium iodide (PI) according to the manufacturer’s protocol [50 (link)] (Beyotime Institute of Biotechnology, Shanghai, China). Cell cycle analysis: after 24 h, we used IE-DAP (10 µg/mL) to treat HepG2 and SMMC-7721 cells for 2 h before exposure to 1 µM Evo for another 24 h. Cells were collected and fixed in 70% ethanol for 24 h at 4 °C, the cell pellet was resuspended in PBS (400 µL), RNaseA (10 mg/mL, 50 µL), and Propidium iodide (PI) (2mg/mL, 10 µL). Mixtures were incubated in the dark at 37 °C for 30 min. Then cell apoptosis rates and cycle were analyzed with FAC-Scan laser flow cytometry (FAC-S, Calibur, Becton Dickinson, Franklin Lakes, NJ, USA). Data was analyzed using CELL Quest software (1.1 version, BD Biosciences, Franklin Lakes, NJ, USA).
Annexin 5 fitc propidium iodide
Manufactured by Beyotime
Sourced in China
Annexin V-FITC/propidium iodide (PI) is a fluorescent staining solution used in flow cytometry applications. Annexin V binds to phosphatidylserine, which is translocated to the outer leaflet of the cell membrane during apoptosis. FITC is a fluorescent label attached to Annexin V. Propidium iodide is a DNA-binding dye that can identify late apoptotic or necrotic cells. This combination of stains allows for the identification and quantification of cells in various stages of cell death.
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7 protocols using annexin 5 fitc propidium iodide
1
Apoptosis and Cell Cycle Analysis of Evo in HepG2 and SMMC-7721 Cells
2
Apoptosis Analysis of MIN6 Cells
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To investigate apoptosis of MIN6 cells, Hoechst 33258 staining (C1017, Beyotime) solution was added to the cells for a final concentration of 10ug/mL and incubated at 37°C for 15min. Cells were collected and chromatin morphologic changes were observed using fluorescence microscopy (Leica, Germany). Reduced nuclear size, chromatin condensation, intense fluorescence and nuclear fragmentation were indicators of apoptotic cells. Then, we further detected apoptosis using flow cytometry. MIN6 cells (5×105) were plated in a 1.5 mL centrifuge tube and washed twice with cold PBS, suspended in binding buffer and stained by Annexin V-FITC/propidium iodide (PI) (C1056, Beyotime). Cells were incubated for 20 min in the dark (37°C) and the FACSCalibur flow cytometer (Acuuri, USA) was used to observe apoptotic cells within 1 hour. Annexin V-FITC+PI−: represented early apoptotic cells, Annexin V-FITC−PI+: represented necrotic cells, Annexin V-FITC+PI+: represented late apoptotic cells necrotic cells and Annexin V-FITC−PI−: represented living cells.
3
Apoptosis Evaluation in HOS Cells
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After HOS cells had been exposed to different treatments for 12 h, their apoptosis rates were detected by flow cytometry after Annexin V-FITC/propidium iodide (PI) (Beyotime Biotech) double staining.
4
Apoptosis Analysis of Caco-2 Cells Treated with miR-4262 Inhibitor
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Caco-2 cell apoptosis was detected via flow cytometric analysis. In the present study, the cells were transfected with miR-4262 inhibitor, inhibitor control, control-siRNA and SIRT1-siRNA for 48 h and treated with 2% DSS for 4 days. Subsequently, the cells were trypsinized at room temperature for 1 min and stained with Annexin V-FITC/propidium iodide (PI; Beyotime Institute of Biotechnology), according to the manufacturer's protocol. The results were analyzed using a FACSCalibur flow cytometer (BD Biosciences) and FlowJo software (version 7.6.1; Tree Star, Inc.).
5
Chondrocyte Apoptosis Analysis
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The apoptotic rate of the chondrocytes (2x105 cells/well) at 80% confluency was analyzed using an apoptosis detection kit [Annexin V-FITC/propidium iodide (PI); Beyotime Institute of Biotechnology] and a BD FACS flow cytometer (BD Biosciences).
6
Quantifying Cell Apoptosis by Flow Cytometry
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The measurement of cell apoptosis was performed using the Annexin V-FITC/propidium iodide (PI) kit (C1062S, Beyotime, China). Cells (5 × 104 per well) were treated according to the above grouping conditions. After treatment, 195 μl binding buffer was added to the cell suspensions. Thereafter, 5 μl Annexin V-FITC and 10 μl PI were added to the cell suspensions and mixed well. The mixture was placed in an incubator at 37°C for 15 min (min). Finally, we used a flow cytometer (DxFLEX, Beckman, USA) to examine the FITC and PI signals.
7
Annexin V-FITC Apoptosis Assay
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Cell apoptosis was analyzed by flow cytometry. Following different treatments, cells were trypsinized and washed with PBS twice. The cells were then stained with Annexin V-FITC/propidium iodide (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. Apoptosis was analyzed by MultiCycle software.
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