Apoptosis-mediated cell death was examined by a double staining method, using a FITC-labeled Annexin V/propidium iodide (PI) apoptosis detection kit (BD Bioscience, San Jose, CA), as previously described [20 ]. Briefly, cells were treated for 24 h and then harvested, washed in cold phosphate-buffered saline (PBS) twice and then stained with fluoresceinisothiocyanate (FITC)-conjugated Annexin V and PI dyes. The externalization of phoshatidylserine and the permeability to PI were evaluated using a FACS Calibur flow cytometer (BD Bioscience). Data from 10,000 gated events per sample were collected. Cells in early stages of apoptosis were positively stained with Annexin V, whereas cells in late apoptosis were positively stained with both Annexin V and PI. Also, to confirm the dependence of apoptosis on caspase activity the test was done with the specific caspase inhibitor, Z-VAD-FMK.
Fitc labeled annexin 5 propidium iodide apoptosis detection kit
Manufactured by BD
Sourced in United States
The FITC-labeled Annexin V/propidium iodide (PI) apoptosis detection kit is a laboratory tool used to identify and quantify apoptotic cells. The kit includes FITC-conjugated Annexin V and propidium iodide, which are commonly used markers for detecting cellular apoptosis.
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4 protocols using fitc labeled annexin 5 propidium iodide apoptosis detection kit
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Annexin V/PI Apoptosis Assay
2
Characterization of Immune Cell Phenotypes
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The cell surfaces was stained with antibodies at 4°C for 30 min. Specific antibodies against CD11b (M1/70), F4/80 (BM8), CD206 (MMR), PD-1 (J43) and PD-L1 (MIH5) were purchased from eBioscience (San Diego, CA). The samples were washed using PBS and fixed with 4% paraformaldehyde. The samples were analyzed using FACSCantoⅡTM flow cytometer (BD Biosciences, San Jose, CA). The data were analyzed using FlowJo software (Tree Star, Ashland, OR). Cell apoptosis was measured using a double staining with FITC-labeled Annexin V/Propidium Iodide (PI) Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s instruction. The apoptosis levels were examined by flow cytometry. For reactive oxygen species (ROS) measurement, the cells were pretreated with 10 mM NAC (Sigma-Aldrich, St. Louis, MO) for 24 h. Then, 10 μM DCF-DA (Sigma-Aldrich) was added to the cells for 30 min at 37°C, and DCF fluorescence was measured by flow cytometry.
3
Detecting Apoptosis in HaCaT Cells
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Apoptosis-mediated death of HaCaT cells was examined by a double staining method using an FITC-labeled annexin V/propidium iodide (PI) apoptosis detection kit (BD Biosciences, San Jose, CA, US) according to the manufacturer’s instructions. In brief, cells were washed twice with phosphate-buffered saline (PBS) and then incubated with PBS containing FITC-conjugated Annexin V and PI at 37°C for 30 min. Cells were washed twice with PBS and then incubated with PBS containing 10 µM Hoechst 33258 at 37°C for 30 min. The externalization of phosphatidylserine and permeability to PI were evaluated with an IN Cell Analyzer 2000 (GE Healthcare UK, Ltd., Buckinghamshire, UK). Cells in early apoptosis were positively stained with Annexin V, whereas cells in late apoptosis were positively stained with both Annexin V and PI.
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Annexin V-PI Apoptosis Assay
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Apoptosis-mediated cell death was examined by a double staining method, using a FITC-labeled Annexin V/propidium iodide (PI) apoptosis detection kit (BD Bioscience, San Jose, CA), as previously described [14 (link)]. Briefly, cells were treated for 12 h and then harvested, washed in cold phosphate-buffered saline (PBS) twice and then stained with fluoresceinisothiocyanate (FITC)-conjugated Annexin V and PI dyes. The externalization of phoshotidylserine and the permeability to PI were evaluated using a FACS Calibur flowcytometer (BD Bioscience). Data from 10,000 gated events per sample were collected. Cells in early stages of apoptosis were positively stained with Annexin V, whereas cells in late apoptosis were positively stained with both Annexin V and PI.
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