Apoptotic cell death was measured using an Annexin V-APC/PI kit (Biolegend, San Diego, CA, USA). A total of 10,000 (cells/well) ASCs were seeded onto 6-well plates and incubated at 37 °C with 5% CO2. The cells were treated with metformin (0.25 mM, 0.5 mM, 1 mM, 2.5 mM, and 5 mM) (Sigma-Aldrich, St. Louis, MO, USA). After 72 h of treatment, the cells were collected by trypsinization and stained with 2.5 μL AnnexinV-APC and 2.5 μL PI in binding buffer, incubated in the dark at room temperature for 15 to 20 min, and analyzed by flow cytometry (Fortessa, BD, NJ, USA).
Annexin 5 apc pi kit
Manufactured by BioLegend
Sourced in United States
The Annexin V-APC PI kit is a laboratory product designed to detect and quantify apoptosis in cell samples. It contains Annexin V conjugated to the fluorescent dye APC and the nuclear stain propidium iodide. This kit allows for the simultaneous detection of early and late apoptotic cells through flow cytometry.
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5 protocols using annexin 5 apc pi kit
1
Metformin-Induced Apoptosis in ASCs
2
Annexin V Apoptosis Assay by Flow Cytometry
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Apoptosis was assessed by Annexin V staining and flow cytometry analysis. Briefly, 5 × 105 SiHa or KoE6/E7 SiHa cells were harvested, washed in PBS, and then analyzed by Annexin V/propidium iodide staining by flow cytometry according to the manufacturer’s protocol (Annexin V-APC PI kit; Biolegend) (Figure S3B
3
Apoptosis Assay using Annexin V-APC/PI
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Apoptosis was determined using the Annexin V-APC PI kit (Biolegend). At 48 h post-transfection, cells were collected by trypsinization and washed with PBS. The cells were resuspended in 300 μl of binding buffer, then 5 μl of Annexin V-APC (stock concentration of 0.5 mg/ml) and 5 μl of PI (stock concentration of 4 mg/ml) were added to the samples for 10 min and incubated in the dark. Apoptosis were determined using CytoFlex and analysed using FlowJo. Statistical analysis was done using GraphPad/Prism. The comparison of means between independent groups was performed using unpaired t-test.
4
EGFR Expression and Apoptosis Analysis
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HBEC-RL53 and H358 cells were washed in PBS containing 1% BSA and 0.1% NaN3 and incubated with saturating concentrations of the EGFR primary antibody (mca1784; Bio-Rad) for 30 min at 4°C. After fixation with paraformaldehyde 3.2%, they were incubated with the Alexa Fluor 647 (Invitrogen) secondary antibody for 30 min and then washed and resuspended in PBS. For apoptosis and cell death analysis, AnnexinV-APC PI kit (BioLegend) was used according to the manufacturer’s instructions. Samples were analyzed by flow cytometry using a FACSCanto II flow cytometer (BD Biosciences). The population of interest was gated according to its FSC/SSC criteria. Data were analyzed with FlowJo software (BD Biosciences). The representative experiment out of three independent experiments was shown.
5
Annexin V-APC PI Apoptosis Assay
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Apoptosis was determined using the Annexin V-APC PI kit (Biolegend). 48 hours after transfection, cells were collected by trypsinization and washed with phosphate buffered saline (PBS). The cells were resuspended in 300 µl of binding buffer, then 5µl of Annexin V-APC (stock concentration of 0.5 mg/ml) and 5µl propidium iodide (stock concentration of 4mg/ml) were added to the samples for 10 minutes and incubated in the dark. Apoptosis were determined using CytoFlex and analysed using FlowJo. Statistical analysis was done using GraphPad/Prism.
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