Apoptosis was evaluated by either annexin V-fluorescein isothiocyanate (FITC) staining (BD Biosciences) or 7-aminoactinomycin D (7-AAD, Sigma–Aldrich) by flow cytometry as described previously [19 (link)].
Annexin 5 fluorescein isothiocyanate fitc staining
Manufactured by BD
Sourced in United States
Annexin V-fluorescein isothiocyanate (FITC) staining is a laboratory technique used to detect and quantify apoptosis, a type of programmed cell death. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine (PS), a phospholipid that is externalized during the early stages of apoptosis. FITC, a fluorescent dye, is conjugated to Annexin V, allowing the visualization and analysis of apoptotic cells using flow cytometry or fluorescence microscopy.
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4 protocols using annexin 5 fluorescein isothiocyanate fitc staining
1
Apoptosis Evaluation by Flow Cytometry
2
Apoptosis and Cell Cycle Analysis
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Apoptosis was examined by Annexin V-fluorescein isothiocyanate (FITC) staining (BD Biosciences, San Jose, CA, U.S.A.) according to the manufacturer’s instructions. Cells were seeded on 6-well plates and incubated for 2 days. Cells were treated with mesoglycan (0.1 μg/ml) for 24 h. The FITC fluorescence intensity of 10,000 cells was measured using a Becton-Dickinson FACS Caliber flow cytometer (BD Biosciences). Cell cycle profiles were analyzed by propidium iodide (PI) staining. A minimum of 10,000 cells in each sample was detected according to intracellular PI fluorescence intensity by flow cytometry, and cell cycle was analyzed by Cell Quest software (BD Biosciences).
3
Apoptosis and Cell Cycle Analysis
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We detached 5 × 105 cells from the plates using 3mM EDTA/PBS and resuspended in 500 μl of ice-cold PBS. Annexin V-fluorescein isothiocyanate (FITC) staining was performed according to the manufacturer’s protocol (BD Pharmingen, San Diego, CA, USA). For cell cycle profile analysis, cells were fixed using 80 % ethanol, stored overnight at 4 °C, and then spun at 1,500 rpm for 5 minutes at 4 °C using a centrifuge (Eppendorf 5810R). Pellets were washed with cold PBS + 1 % serum, mixed, spun for 5 minutes at 1,200 rpm, and stained with propium iodide (PI)/RNase solution (BD Pharmingen). All analysis was performed using FACS Diva software on a BD FACSCanto II flow cytometer.
4
Apoptosis Assays in VSMC
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Apoptosis was examined by Annexin V-fluorescein isothiocyanate (FITC) staining (BD Biosciences, San Jose, CA, USA). After seeding of VSMCs (3 × 105) on 6-well plates, cells were treated with H2O2, quercetin and/or compound C. The FITC fluorescence intensity of 10,000 cells was measured using a Becton-Dickinson FACS Caliber flow cytometer (BD Biosciences). Additionally, analysis by acridine orange (AO) staining was used to examine the apoptosis. Cells grown in 6-well plates were washed with PBS and then incubated with AO solution (Immuno Chemistry Technologies, Bloomington, MN, USA) for 20 min at 37℃. After incubation, cells were washed with PBS and imaged under a fluorescence microscope (Olympus, Tokyo, Japan).
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