Ir live dead fixable dead cell stains

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.

Nuclei were isolated from liquid nitrogen flash frozen PDOX tumors [92 (link)]. Samples were minced in DAPI buffer [10 μg/ml DAPI in 146 mM NaCl, 10 mM Tris–HCl (pH 7.5), 0.2% IPEGAL]. Nuclei were disaggregated subsequently with 20G and 25G needles and filtered through a 50 μm and a 30 μm mesh. Tumor nuclei were stained with the human-specific anti-Lamin A/C-PE antibody (Supplementary Table 2, online resource). Optionally, PDOX-derived single cell tumor cells and cell lines were stained with IR-LIVE/DEAD® Fixable Dead Cell Stains (Invitrogen; 1 µg/ml) and fixed with cold 80% ethanol. PBMCs were added to each sample as internal diploid control. Flow analysis was carried out with Aria^TMSORP or Canto^TM flow cytometers (BD Biosystems). DNA content was analyzed with the FlowJo software.