Annexin 5 fluorescein isothiocyanate fitc 7 amino actinomycin d 7 aad staining system

Apoptosis was analyzed with an annexin V-fluorescein isothiocyanate (FITC)/7-amino-actinomycin D (7-AAD) staining system (BD Biosciences) and a Cell Lab Quanta™ SC MPL (Beckman Coulter). Cells were stained with annexin V-FITC only (early apoptotic) or both annexin V-FITC and 7-AAD (late apoptotic) were considered as the apoptotic cell fractions.

Cells were harvested 48 h after culturing in FBS-free medium, washed twice with PBS, and were resuspended in cold binding buffer. For apoptosis analysis, cells were stained with the Annexin V-fluorescein isothiocyanate (FITC)/7-amino-actinomycin D (7-AAD) staining system obtained from BD Biosciences (San Diego, CA, USA). For each assay, 1 × 10⁵ cells were incubated with 5 μL of Annexin V-FITC and 5 μL of 7-AAD in the dark for 15 min at room temperature. After adding 400 μL of binding buffer, the cells were analyzed within one hour by fluorescence-activated cell sorting using FACScan (BD Biosciences, San Diego, CA, USA). Cell populations were classified as viable (Annexin V negative, 7-AAD negative), apoptotic (Annexin V positive, 7-AAD negative or positive), or necrotic (Annexin V negative, 7-AAD positive). All samples were assayed in triplicate.

Fluorescence-activated cell-sorting (FACS) analysis for apoptosis was done 48 hours post-transfection using an annexin V-fluorescein isothiocyanate (FITC)/7-amino-actinomycin D (7-AAD) staining system obtained from BD Biosciences (San Jose, CA) and a BD FACSVerse^™ flow cytometer (BD Biosciences). Cells were stained with annexin V-FITC only (early apoptotic) or both annexin V-FITC and 7-AAD (late apoptotic), and the combined sum was considered to be the total apoptotic cell fraction.