This protocol describes a strategy for lentiviral infection of primary patient pancreatic cancer cells to assess the impact of gene modulation on tumor cell growth in xenografts.
A protocol for lentiviral infection of primary patient pancreatic tumors
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posted 20 Jun, 2016
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Pancreatic cancer is now the 3rd leading cause of cancer related deaths in the United States, and is projected to become the 2nd leading cause by 2020. A better understanding of the molecular regulation of pancreatic cancer growth may help identify new therapeutic strategies targeting this largely drug resistant disease. The development of methods to successfully deliver shRNAs to block gene expression in primary patient pancreatic tumor cells can be critical to defining the networks that control human tumor cell growth.
• Forceps (FST)
• Surgical Scissors (FST)
• Betadine (Fisher)
• Isoflurane (Piramal Healthcare)
• RPMI 1640 (Gibco, Life Technologies)
• Fetal Bovine Serum (Gemini Bio-Products)
• NEAA, Pen/Strep, and Glutamax (all 100x from Gibco, Life Technologies)
• FACS Antibody: EpCAM (PE) antibody was purchased from eBioscience.
• RBC Lysis Buffer (eBioscience)
• Polybrene (Sigma, 5ug/ml)
• Human Tumor Dissociation Kit (Miltenyi, 130-095-929)
• gentleMACS C-tubes (Miltenyi, 130-093-237)
• Soybean Trypsin Inhibitor (Sigma)
• BD Matrigel Growth Factor Reduced (BD Biosciences, 354230)
• Ultra-Low attachment Flat bottom 24 well plates (Corning)
• Isoflurane regulator and scavenging system (South Coast Anesthesia)
• gentleMACS Dissociator (Miltenyi, 130-093-235)
• MACSmix Tube rotator (Miltenyi, 130-090-753)
• FACSAria III machine (Becton Dickinson)
(the procedures below are performed on mice bearing patient-derived pancreatic tumors)
Lentiviral infection Protocol.