Flow Cytometry Analysis of Xenograft Cells

Flow cytometry experiments were performed as described before [16 (link), 17 (link)]. Briefly, xenografts derived in eGFP-expressing mice were minced with scalpels and dissociated with MACS Neural Tissue Dissociation Kit (P) (Miltenyi, 130-092-628, Lund, Sweden) following the manufacturer’s instructions. Single cell suspensions were incubated with Hoechst 33342 (5 µg/ml, Bisbenzimide, Ho342; Sigma) at 37 °C in pre-warmed DMEM, containing 2 % FBS, 10 mM HEPES pH 7.4 and DNAse I (10 µg/ml; Sigma) at 1 × 10⁶ cells/ml for 120 min. After washing, cells were resuspended in ice-cold HBSS 2 % FBS and 10 mM HEPES pH 7.4 buffer (100 µl/test). Prior to flow cytometry, cells were incubated with LIVE/DEAD^® Fixable Dead Cell Stains (Life Technologies) and appropriate preconjugated antibodies for 30 min at 4 °C in the dark (antibodies are listed in Supplementary Table II). Data acquisition was performed on a FACS Aria™ SORP cytometer (BD Biosciences, San Jose, CA, USA) and the Hoechst signal was excited with the UV laser. Data acquisition and analysis were done with DIVA software (BD Biosciences). Histograms were prepared with the FlowJo software.