Single-Cell Analysis of Xenografts

Cell cultures were dissociated using Accutase® (Sigma-Aldrich). Xenografts were dissociated with MACS Neural Tissue Dissociation Kit (P) (Miltenyi) following the manufacturers’ instructions. Single cells were resuspended in HBSS, 2% FBS, 10 mM HEPES buffer (100 µl per test). Cells were incubated with the IR-LIVE/DEAD® Fixable Dead Cell Stains (Invitrogen; 1 µg ml⁻¹) and appropriate preconjugated antibodies for 30 min at 4 °C in the dark (Supplementary Table 3). For cell cycle analysis in viable cells, cells were prestained with Hoechst 33342 (5 µg ml⁻¹, Bisbenzimide, Ho342; Sigma) at 37 °C before antibody staining^{37 (link)}. Data acquisition was performed on a FACS Aria^TM SORP cytometer (BD Biosciences) and ImageStream imaging cytometer (Amnis). Data acquisition and analysis were done for FACSAria with DIVA software (BD Bioscience); and INSPIRE and IDEAS^® for ImageStream. Histograms were prepared with the FlowJo software.