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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