Engineered Cell Lines for Immunotherapy Research

GL261 were provided by A. Fontana, Experimental Immunology, University of Zurich, Zurich, Switzerland and cultured in DMEM (Gibco) supplemented with 10% FCS (Biosera), GlutaMAX (Gibco), and sodium pyruvate 1 mM (Gibco). B16.F10 were bought from ATCC and cultured in RPMI (Gibco) supplemented with 10% FCS (Biosera) and GlutaMAX (Gibco). To express EGFRvIII or GD2, cells were transduced in six-well plates with the addition of 3 mL of a γ-retroviral vector in the presence of 10 µg/mL Polybrene (Sigma). The γ-retroviral vector was produced by transient triple transfection of HEK293T using GeneJuice transfection reagent (Merck Millipore) with 4.69 μg of Peq-Pam plasmid (encoding Moloney GagPol), 3.13 μg of VSV-G envelope, and 4.69 μg of retroviral backbone SFG expressing the gene of interest, EGFRvIII or GD2 and GD3 synthase^{55 (link)} (AddGene plasmid #75013), respectively. Supernatants containing retroviral vector were collected at 48 and 72 h post-transfection and frozen at −80 °C prior to use for GL261 or B16.F10 transduction.