Live/Dead Staining of ASCs and DFATs

2D cultures and 3D spheroids of ASCs and DFATs were simultaneously stained with the green Calcein AM (Cal-AM, 0.25 µL/mL) dye and red ethidium homodimer-1 (1 µL/mL, EthD-1) dye (Invitrogen). The cells were incubated with a mix of dyes for 30 min in darkness at room temperature and then observed and photographed under fluorescence microscopy Axio Vert.A1 (Carl Zeiss, Oberkochen, Germany). To obtain the precise number of living and dead cells, the 3D spheroids were firstly disrupted with Accutase Cell Detachment (Beckton Dickinson) for 15 min, then stained with Cal-AM + EthD-1 and Hoechst 33342 dye (1 µg/mL, Sigma-Aldrich) for the nuclei staining. Live and dead ASCs and DFATs in both 2D and 3D forms were counted with the ZEN 2 Blue Edition software (Carl Zeiss, Oberkochen, Germany), according to the previously described protocols [11 (link)].