Apoptosis and Necrosis Analysis of GMSC Spheroids

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 instructions^{18 (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 FACSCanto^TM II flow cytometer (BD Bioscience).