The apoptosis assay was performed using the fluorescein isothiocyanate (FITC) Annexin V apoptosis detection kit with propidium iodide (BioLegend, San Diego, CA, USA) according to manufacturer’s protocol. Cells were collected and re-suspended in 1× binding buffer. Double staining was done by incubating the cell suspension with Annexin V-FITC and propidium iodide solution for 15 min at RT in the dark. Annexin V (FL1) and propidium iodide (FL3) fluorescence were measured with a FACS FC500 System flow cytometer (Beckman Coulter South Africa (Pty) Ltd., Johannesburg, South Africa) equipped with an air-cooled argon laser excited at 488 nm. Results were expressed in percentage of cells in three categories, namely viable cells, apoptotic cells and necrotic cells.
Annexin 5 apoptosis detection kit with propidium iodide
Manufactured by BioLegend
Sourced in United States
The Annexin V Apoptosis Detection Kit with propidium iodide (PI) is a laboratory tool used for the detection and analysis of apoptosis (programmed cell death) in cell samples. The kit utilizes Annexin V, a protein that binds to phosphatidylserine, a lipid that is exposed on the surface of apoptotic cells. The kit also includes propidium iodide, a fluorescent dye that stains the DNA of cells with compromised cell membranes, typically associated with late-stage apoptosis or necrosis. The kit allows for the identification and quantification of cells undergoing various stages of apoptosis through flow cytometry or fluorescence microscopy analysis.
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5 protocols using annexin 5 apoptosis detection kit with propidium iodide
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Annexin V-FITC Apoptosis Assay
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Docetaxel-Induced Apoptosis Analysis
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Tumor cells were treated with 20 ng mL−1 docetaxel for 12 h and digested with 0.25% trypsin. Apoptosis was detected using the Annexin V Apoptosis Detection Kit with propidium iodide (Cat# 640 932, Biolegend) following the manufacturer's instructions. Briefly, cells were washed with PBS, and then incubated with 100 µL of Annexin V binding buffer containing 5 µL of APC‐conjugated Annexin V and 10 µL of propidium iodide solution for 15 min at 26 °C. Next, 400 µL of Annexin V Binding Buffer was added to each sample, and the cells were immediately analyzed by flow cytometry.
3
Apoptosis and Cell Cycle Analysis
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Apoptosis was assessed using the Annexin V Apoptosis Detection Kit with propidium iodide (PI) (Biolegend Pacific Blue™) according to the manufacturer’s recommendations. For cell cycle analysis, DNA was stained with PI and cells were analysed using a BD LSRII flow cytometer. The percentage of cells in each stage of the cell cycle was then determined using the ModFit software (Verity Software House).
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Evaluating Apoptosis in NPC Cells Exposed to γδ-T-Exos
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NP69, HK-1, NPC43 or C666-1 cells were treated with PBS, γδ-T-Exos, γδ-T-Exos-depleted conditioned medium, or the ultracentrifuged pellets from non-conditioned FBS–exosome-free medium without γδ-T-cell components. Eighteen or 24 hours later, the cell apoptosis was detected using an Annexin V Apoptosis Detection Kit with propidium iodide (PI; BioLegend) as we did before.38 (link) In some experiments, γδ-T-Exos were stained with CFSE before incubation with NPC cells, and the cell death was determined at the indicated time. In some experiments, γδ-T-Exos were preincubated with blocking anti-FasL, anti-TRAIL antibodies or isotype control (BioLegend). In some experiments, activated caspase-3 was detected in permeabilized NPC cells after 4-hour exposure to γδ-T-Exos using an antiactive-caspase-3 monoclonal antibody (BD Pharmingen).39 (link) In some experiments, NPC cells were irradiated at 0, 1, or 3 Gy before treatment with γδ-T-Exos; then the cell apoptosis was determined as described earlier. In some experiments, NPC cells were treated with γδ-T-Exos or PBS in the presence or absence of 50% NPC supernatant.
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Annexin V-PI Apoptosis Assay
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For apoptosis assessment the Annexin V Apoptosis Detection kit with Propidium Iodide (PI) (Biolegend) was used according to the manufacturer’s instructions and BMDMs were measured using a LSR II flow cytometer. First, single cells were gated. PI + cells were identified as dead cells, Annexin V + cells were identified as apoptotic cells. The remaining cells were identified as live cells.
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