Cell apoptosis was measured using an Annexin V/PI staining assay. The cells were seeded at a density of 2×106 cells in a 6-well plate, and when the cells reached 80% confluence they were treated with different doses of PAB (2.5–10 µM) and irradiated with 4 Gy. After 24 h the cells were washed with PBS, collected and then suspended in a Sigma-Aldrich Annexin V binding buffer (Sigma-Aldrich; Merck KGaA) according to the manufacturer's instructions. Annexin V and PI (Sigma-Aldrich; Merck KGaA) were added to the sample and it was incubated for 30 min at 37°C. The cell apoptosis rate was analyzed using a flow cytometer (BD Biosciences). Autophagy ratios were analyzed by CellQuest software (version 3; BD Biosciences).
Annexin 5 and pi
Manufactured by Merck Group
Sourced in United States
Annexin-V and PI are laboratory reagents used for the detection and analysis of apoptosis, a programmed cell death process. Annexin-V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, which is exposed on the cell surface during apoptosis. PI (Propidium Iodide) is a fluorescent dye that binds to DNA and is used to identify cells with disrupted cell membranes, typically associated with late-stage apoptosis or necrosis. These reagents are commonly used in flow cytometry and other cell analysis techniques to assess the degree of apoptosis in a cell population.
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6 protocols using annexin 5 and pi
1
Apoptosis and Autophagy Analysis
2
Apoptosis Quantification in HCC Cells
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After various treatments, HCC cells were washed twice with 1× PBS and centrifuged. Then, 100 μl of FITC-conjugated Annexin-V and PI (Sigma, USA) was added and the cells were incubated for 20 min at room temperature in the dark. Then, the cells were subjected to flow cytometric analysis (Merck Millipore, Germany). The percentage of apoptotic cells (Annexin V+ PI+) was determined by using FlowJo software 7.6 (Treestar, USA).
3
Modulation of Oxaliplatin-Induced Apoptosis
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After transfection with miR-29b, NCO and SIRT1 plasmid, OR-SW480 cells were treated with oxaliplatin (10 μM) for 48 h. Subsequently, cells were collected and washed with PBS. For apoptosis measurement, cells were incubated with Annexin V and PI (Sigma Aldrich, USA) followed by analyzing on flow cytometry. For evaluation of ROS production, cells were stained with dihydroethidium (DHE) (Molecular Probes, USA) followed by analyzing on flow cytometry. The non-overlapping area normalized to the NCO group was represented as the generated ROS in the experimental groups.
4
Annexin-V Apoptosis Assay in MDA-MB-231 Cells
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Annexin-V binding is indicative of early apoptosis. MDA-MB-231 (2×105cells/ml) were cultured in six-well plates treated with 2 μM JA for 48 h, harvested and washed with phosphate-buffered saline (PBS). Both the adherent and non-adherent cell fractions were probed with fluorescein isothiocyanate (FITC)-conjugated Annexin-V and PI (Sigma, St. Louis, MO) for 15 min. The staining profiles were determined with FACScan and Cell-Quest software. The experiments were performed two times.
5
Annexin V and PI Apoptosis Assay
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An annexin V and PI (Sigma-Aldrich) stain was performed to determine the apoptosis levels after eribulin or abemaciclib treatment. All cells were harvested 48 h after drug treatment, washed in PBS, and resuspended with 400 µL annexin V 1 × binding buffer. Cells were stained with annexin V-APC and PI and incubated for 15 min at room temperature in the dark. Stained cells were washed twice with PBS and analyzed for apoptosis with flow cytometry. Moreover, 10,000 events were recorded using flow cytometry (Beckman Coulter CytoFLEX), and the proportion of apoptotic cells was analyzed.
6
Apoptosis Quantification by Flow Cytometry
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Cells with transfections were washed with PBS (ice-cold), and staining in dark with FITC labeled Annexin-V and PI (Sigma-Aldrich) was performed 15 min. After that, flow cytometry was used to separate apoptotic cells. The group with the highest apoptotic cell percentage was set to ‘100%’, and all other groups were normalized to this group. Three independent replicates were set for each experiment.
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