Generating Luciferase-Expressing Mouse Pancreatic Tumor Cells

Pan02 cells, a SMAD4-null chemically induced mouse pancreatic tumour line, was from the National Cancer Institute [45 (link), 46 (link)]. Pan02-CAG-luc2 cells were generated by lentiviral transduction. Briefly, DNA encoding the luciferase 2 gene (luc2, Promega) was cloned into a lentiviral vector (pCDH-CAG-IRES-puro, System Biosciences). Lentivirus was generated using the pPack lentiviral packaging system (SBI), and used to transduce Pan02 cells. Following antibiotic selection, a pool of stably transduced cells (Pan02-CAG-luc2) was obtained and used for in vivo studies. Luc2 expression in the Pan02-CAG-luc2 cell line pool was stable up to passage 16, as determined by luciferase assay (Promega, data not shown).
The following antibodies from eBioscience, BD Bioscience and Biolegend were used for flow cytometry: anti-CD45 (clone 30-F11), anti-CD44 (clone IM7), anti-CD62L (clone MEL-14), anti-CD3 (clone 145-2C11), anti-CD4 (Clone RM4.5), anti-CD8 (clone 53-6.7), anti-F4/80 (clone BM8), anti-MHCI (clone 28-8-6), anti-CD80 (clone 16-10A), anti-CD86 (clone GL-1), anti-MHCII (clone M5/114.15.2), anti-Gr-1 (clone RB6-8C5), anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-CD19 (clone6D5), anti-PD-L1 (clone 10F.9G2), anti-PD-1 (clone 29F.1A12), anti-FoxP3 (FJK-16S).

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Luheshi N.M., Coates-Ulrichsen J., Harper J., Mullins S., Sulikowski M.G., Martin P., Brown L., Lewis A., Davies G., Morrow M, & Wilkinson R.W. (2016). Transformation of the tumour microenvironment by a CD40 agonist antibody correlates with improved responses to PD-L1 blockade in a mouse orthotopic pancreatic tumour model. Oncotarget, 7(14), 18508-18520.

Publication 2016

luciferase assay anti-CD4 anti-Gr-1 anti-PD-L1 anti-PD-1

Anti cd3 Anti cd80 Antibiotic Antibodies Assay Cd11b Cd44 Cd62l Cell line Cells Clone Flow cytometry Gene Ires Lentivirus Luciferase Null mouse Pancreatic tumour Pd l1 Ppack Promega Smad4 Vector

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Variable analysis

independent variables

Lentiviral transduction of Pan02 cells with DNA encoding the luciferase 2 gene (luc2)

dependent variables

Luciferase expression in the Pan02-CAG-luc2 cell line pool
Stability of luc2 expression in the Pan02-CAG-luc2 cell line pool up to passage 16

control variables

Pan02 cells, a SMAD4-null chemically induced mouse pancreatic tumour line
Antibiotic selection after lentiviral transduction to obtain a pool of stably transduced cells (Pan02-CAG-luc2)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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