Plenti pgk v5 luc neo

The AT-3 tumor cell line was established from a primary mammary gland carcinoma of the PyMT-MMTV transgenic mice on a B6 strain and was maintained as described^{31 (link)}. AT-3 cells were cultured in Gibco Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) (Sigma), 1% non-essential amino acid (NEAA) (Gibco), 2 mM l-glutamine (Gibco), 0.5% penicillin/streptomycin (Gibco), and 55 μM 2-mercaptoethanol (Gibco). AT-3 tumor cells expressing luciferase (AT-3-luc) were generated with infection of lentiviruses encoding luciferase (pLenti PGK V5-LUC Neo, Addgene plasmid #21471). These cell lines were authenticated by morphology, phenotype, and growth, and routinely screened for Mycoplasma, and were maintained at 37 °C in a humidified 7% CO2 atmosphere.

The AT-3 tumor cell line was established from a primary mammary gland carcinoma of the PyMT-MMTV transgenic mice on a B6 strain and was maintained as described^{66 (link)}. The 4T1 breast cancer and B16-F10 (B16) melanoma cell lines were purchased from the American Type Culture Collection (ATCC), authenticated at ATCC, and maintained. The MC38 colon adenocarcinoma cell line was gift from Dr. Weiping Zou (University of Michigan). AT-3 cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Sigma), 1% NEAA (Gibco), 2 mM l-glutamine (Gibco), 0.5% penicillin/streptomycin (Gibco), and 55 μM 2-mercaptoethanol (Gibco). 4T1, MC38, and B16 cells were cultured in RPMI (Gibco) supplemented with 10% FBS, 1% NEAA, 2 mM l-glutamine, 0.5% penicillin/streptomycin, and 55 μM 2-mercaptoethanol. For generation of mouse AT-3 cells expressing GFP (AT-3-GFP), AT-3 cells were infected with retroviruses encoding GFP (mEGFP-N1, Addgene plasmid #54767 a gift from Dr. Michael Davidson). 4T1 cell expressing Luciferase (4T1-luc) were generated with infection of lentiviruses encoding Luciferase (pLenti PGK V5-LUC Neo, Addgene plasmid #21471). These cell lines were authenticated by morphology, phenotype, and growth, and routinely screened for Mycoplasma, and were maintained at 37 °C in a humidified 5% CO₂ atmosphere.

Murine Lewis lung carcinoma (LLC) cells were purchased from ATCC^® (American Type Culture Collection, Manassas, VA, USA) and cultured in either T-75 or T-175 flasks. Cells were passaged for subculturing by first aspirating the culture medium with a pipette, rinsing with 1× phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA, SH30256FS), aspirating off the PBS, then rinsing with 0.25% trypsin-0.53 mM EDTA solution (Thermo Fisher Scientific, Waltham, MA, USA, 25-200-056), and then they were neutralized with complete growth media consisting of Dulbecco’s modified Eagle’s medium (DMEM, ATCC^®, Manassas, VA, USA) with 10% fetal bovine serum (FBS, USDA approved, ATCC^®, Manassas, VA, USA), and 1% Penicillin-Streptomycin (10,000 U/mL, Thermo Fisher Scientific, Waltham, MA, USA). Cells were modified to be luciferase-expressing (LLC-Luc), as previously described [54 (link)] through use of plasmid pLenti PGK V5-LUC Neo [63 (link)] (Addgene, Cambridge, MA, USA) which was packaged in lentiviral particles and performed at the Baylor College of Medicine (BCM) vector core facility. For the luciferase-expressing cells, 1% Geneticin (Thermo Fisher Scientific, Waltham, MA, USA) was added to the media to maintain culture. Cells were maintained in a HERAcell 150i CO2 incubator (Thermo Fisher Scientific, Waltham, MA, USA) set to 37 °C and 5% humidity.

F98 GBM cells (kindly provided by Prof. C. Vanhove, Ghent University, Belgium) were cultured in DMEM (Dulbecco’s modified eagle medium) supplemented with 10% fetal bovine serum (Sigma-Aldrich, Zwijndrecht, Netherlands). Cells were transduced with a lentiviral vector harboring a PGK-driven FLuc+ (pLenti PGK V5-LUC neo (w623-2), a gift from Eric Campeau (Addgene plasmid #21471; http://n2t.net/addgene:21471; RRID:Addgene_21471)) [40 (link)]. Neomycin selection began 48 h after transduction and after three weeks of continuous selection, cells could be used in experiments. Cells were tested for the presence of mycoplasma prior to injection into animals.