The Annexin-V and propidium iodide (PI) staining kit (BD bioscience) was used to assess cell death according to the manufacturer’s protocol. In brief, cells were seeded into a 6-well plate at a density of 1 × 106 cells/well. After different treatments, cells were collected and stained with 5 μL of FITC-Annexin V and PI solution for 30 min at room temperature, in the dark. The number of positively stained cells was measured using FACSCalibur (BD Bioscience, USA). Caspase cleavage was assessed as an additional indicator of apoptosis.
Annexin 5 and propidium iodide staining kit
Manufactured by BD
The Annexin-V and propidium iodide (PI) staining kit is a laboratory tool used to detect and quantify apoptosis, a form of programmed cell death. Annexin-V is a protein that binds to phosphatidylserine, a lipid that is externalized on the surface of cells undergoing apoptosis. Propidium iodide (PI) is a dye that binds to the DNA of cells with compromised cell membranes, indicating late-stage apoptosis or necrosis. The kit provides the reagents necessary to perform this assay, allowing researchers to analyze the progression of cell death using flow cytometry or fluorescence microscopy.
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2 protocols using annexin 5 and propidium iodide staining kit
1
Annexin-V and PI Staining for Cell Death
2
Measuring Cell Viability in hMDMs and Adipocytes
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For hMDMs and non-adherent PBMCs, cell viability was measured using the Annexin V and propidium iodide (PI) staining kit (BD Biosciences) according to the manufacturer's instructions. hMDMs were detached using non-enzymatic cell dissociation buffer (Sigma) and 5x10 4 cells were resuspended with annexin V binding buffer. hMDMs and non-adherent PBMCs were stained with annexin V (25 µg/ml) and PI (125 ng/ml) and fluorescent cells were detected using a Fortessa X20 (BD Biosciences) flow cytometer. The results were analysed using the FlowJo (v10.6) software. Duplets were excluded by gating FSC area versus FSC height and AnnexinV -PI -cells were considered viable cells.
For WAT-derived adipocytes, viability was measured by adding AlamarBlue (Thermofisher) directly into the culture (10%) for 4 hours. Light absorbance was measured using Multiscan Ascent (Thermo Fisher). The percentage of reduced AlamarBlue and cell viability were quantified following manufacturer's instructions.
For WAT-derived adipocytes, viability was measured by adding AlamarBlue (Thermofisher) directly into the culture (10%) for 4 hours. Light absorbance was measured using Multiscan Ascent (Thermo Fisher). The percentage of reduced AlamarBlue and cell viability were quantified following manufacturer's instructions.
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