Apoptosis and necrosis in cell mixtures containing 0 – 100% drug-treated cells were visualized by flow cytometry using a method adapted from (13 (link)), in parallel to cell uptake studies. Fluorescein isothiocyanate (FITC)-Annexin V (BioLegend) in combination with 7-amino-antinomycin D (7-AAD; BioLegend) were used for cell death determination. Early apoptotic cells were defined as cells positively stained for FITC-Annexin V but not 7-AAD, with both late apoptotic and necrotic classified as cells positive for both stains. FlowJo (v.7.6.5, Tree Star, Inc.) was used for analysis.
7 amino antinomycin d 7 aad
Manufactured by BioLegend
7-amino-antinomycin D (7-AAD) is a fluorescent dye used for flow cytometry applications. It is a cell-impermeant dye that binds to DNA, allowing for the identification of dead cells in a sample.
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2 protocols using 7 amino antinomycin d 7 aad
1
Apoptosis and Necrosis Quantification
2
Apoptosis and Necrosis Analysis of GMSC Spheroids
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Apoptosis and necrosis of GMSC spheroids were detected using the Fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection kit with 7-amino-antinomycin D (7-AAD) (BioLegend) according to the manufacturer’s instructions18 (link). Briefly, GMSC spheroids were dissociated with StemPro® Accutase® Cell Dissociation Reagent (STEMCELLS Technologies, Inc.) and the dissociated single cells were resuspended in 100 μl of Annexin V binding buffer with a cell density of about 5 × 106 cells/ml and mixed with 5 μl of FITC-conjugated Annexin V and 7-AAD. Following incubation at room temperature for 15 min in the dark, 400 μl of Annexin V binding buffer was added and then the stained cells were analyzed using a BD FACSCantoTM II flow cytometer (BD Bioscience).
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