Ttc 466

ES cell lines (A673, RD-ES, SKES-1, SK-N-MC, TC-32 and TTC-466) were cultured as previously described [23 (link)] and purchased from the American Type Culture Collection, Manassas, VA, except for the following cells that were kind gifts: TC-32 cells from Dr. J. Toretsky (Division of Pediatrics, University of Maryland, Baltimore, MD, USA), the TTC-466 cells from Dr. P. Sorenson (British Columbia Children’s Hospital, Vancouver, BC, Canada). Primary ES cell cultures and daughter ES-CSCs were cultured as previously described [17 (link)]. The embryonic stem cell (ESC) culture SHEF-4 (RRID:CVCL_9791) was a gift from Professor P. Andrews, Centre for Stem Cell Biology, University of Sheffield, Sheffield, UK [24 (link)] and used as a positive control for CD133 and ATP binding cassette subfamily G member 1 (ABCG1). The glioblastoma cell line (T98G) was a gift from Professor M. Knowles, University of Leeds, and was a positive control for multidrug resistance-associated protein 1 (MRP1, gene name = ABCC1) [23 (link)]. The human embryonic fibroblast line KMST-6 (cultured in MEM containing 10% FBS and 2 mM glutamine) and Jurkat cells (cultured in RPMI 1640 containing 10% FBS and 2 mM glutamine) were gifts from Dr E. Morrison, University of Leeds, and used as the positive control and to generate the calibration curve, respectively, for the measurement of telomere length.

Ewing sarcoma cell lines (n = 21) were obtained from multiple sources: L-1062 and L-872 were established in-house [21 (link)]; CHP100, RM-82, IARC-EW7, TC32 and 6647, CHP100, RM-82, IARC-EW-7, WE-68, IARC-EW-3, STA-ET-2.1, TTC-466, STA-ET-10, CADO-ES1, TC-71, VH-64, COH and STA-ET-1 were obtained from the EuroBoNeT consortium collection (Institute of Pathology, University Medical Center, Düsseldorf, Germany) [22 (link)] and SK-ES-1, SK-NM-C, A-673 and R-D-ES from the American Type Culture Collection (ATCC). All cell lines and primary culture L-4027 were cultured in a monolayer under equal conditions and in Iscove’s modified Dulbecco’s medium containing GlutaMAX supplement, supplemented with 1 % streptomycin/penicillin and 10 % heat-inactivated FCS (all from Life Technologies, Bleiswijk, The Netherlands). Authentication of cell lines using Powerplex 1.2 and CellID STR (Promega, Leiden, the Netherlands) and mycoplasma DNA Q-PCR screening were regularly performed on all cell lines.