Ex Vivo T Cell Stimulation in the Pancreatic Tumor: A Method to Increase Cytokine Production in T Cells
Published: April 30, 2023
Abstract
Source: Spear, S., et al. Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity. J. Vis. Exp.(2019).
This article describes the technique for ex vivo T cell stimulation to increase the intracellular concentration of cytokines such as interferon-gamma or IFN-gamma and tumor necrosis factor-alpha or TNF-alpha in the T cell. These cytokines mediate an effective anti-tumor response and can have a therapeutic role in the tumor.
Protocol
1. Digestion of pancreatic tumors Place the dissected pancreatic tumor, metastatic site tumors, or healthy pancreatic tissue in ice-cold PBS, and store on ice. Use forceps to transfer the tumor onto a Petri dish. Add 5.0 mL of digestion medium (2 mg/mL Collagenase, 0.025 mg/mL DNase RPMI) into a 50 mL tube; store on ice to prevent enzyme activity commencing. NOTE: This protocol uses Collagenase Type V, which has an activity of ≥1 units/mg FALGPA and > …
Divulgazioni
The authors have nothing to disclose.
Materials
| 70 μm pore-size cell strainer | Fisher Scientific | 11597522 | |
| anti-CD107a PE (clone 1D4B) | Biolegend | 121612 | 1:100 to culture media |
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A4503 | |
| Cell Stimulation Cocktail (500x) (phorbol 12-myristate 13-acetate (PMA) and ionomycin) | eBioscience | 00-4970-03 | x Final concentration PMA 0.081 μM, ionomycin 1.34 μM |
| THP1 Human Monocytic Leukemia Cells | ATCC | TIB-202 | |
| Collagenase Type V from Clostridium histolyticum | Sigma-Aldrich | C9263 | 2 mg/mL in media |
| Dimethyl sulphoxide (DMSO) | Sigma-Aldrich | D2650-100mL | |
| DNase (Deoxyribonuclease I from bovine pancreas Type II-S) stock 10 mg/mL in 0.15 M NaCl | Sigma-Aldrich | D4513 | Final concentration in digestion media 0.025 mg/mL |
| Foetal calf-serum (FCS) | GE Healthcare | A15-104 | 10% in RPMI |
| Hamilton syringe 700 series, 25 μL volume, 22s gauge needle bevel tip | Fisher Scientific | 10100332 | |
| Penicillin/streptomycin | PAA | 15140122 | 100 units/mL Penicillin, 100 μg/mL Streptomycin |
| Protein transport inhibitor cocktail (500x) (brefeldin A and monesin) | eBioscience | 00-4980-03 | 1x Final concentration Brefeldin A 10.6 μM, monensin 2 μM |
| RPMI-1640 (containing 0.3 g/L Glutamine) | Sigma-Aldrich | R8758 | |
| Surgical Scalpel Blade No.10 | Swann-Morton | 501 | |
| Trypsin-EDTA Solution 10x | Sigma-Aldrich | 594-18C | Trypsin (0.1%) EDTA (0.4%) final concentration |
| U-bottomed 96 microwell plate | VWR | 734-2080 |
Citazione di questo articolo
Ex Vivo T Cell Stimulation in the Pancreatic Tumor: A Method to Increase Cytokine Production in T Cells. J. Vis. Exp. (Pending Publication), e20306, doi: (2023).