Encyclopedia of Experiments: Cancer Research
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Transcript
- Activated T cells move to the pancreatic tumor microenvironment, where they release cytokines like interferon-gamma, or IFN gamma, and tumor necrosis factor-alpha, or TNF alpha, which helps mediate an effective antitumor response. As cytokines rapidly move out of the T cell, the intracellular levels can be undetectable. Therefore, the T cell requires stimulation to increase basal cytokine production for studying their therapeutic role in the tumor.
Begin by taking a suspension of tumor cells from the mouse's pancreas. Now, treat the T cells present in cell suspension with a cytokine stimulation cocktail and incubate the mix for desired time. This cocktail facilitates the release of calcium ions from the endoplasmic reticulum to the cytoplasm. High calcium levels, in turn, activate certain nuclear transcription factors, leading to an increase in cytokine production in the T cells.
Next, treat the cells with a protein transport inhibitor cocktail and incubate the tube. This cocktail blocks cytokine transport from the endoplasmic reticulum to the golgi complex, resulting in the accumulation of cytokines within the cell. Finally, stain the cells using appropriate intracellular markers for flow cytometry analysis. In the following protocol, we will perform T cell stimulation to increase the intracellular concentration of cytokines.
- To begin, use forceps to transfer the tumor onto a Petri dish. Store 5 milliliters of digestion medium in a 50-milliliter tube on ice to prevent enzyme activity commencing. Take a small aliquot of this solution to cover the tumor on the Petri dish. Use a sterile scalpel and forceps to cut the tumor into small pieces, roughly less than 3 millimeters in length. Scrape the tumor pieces into the tube and gently invert the tube until all pieces are submerged in digestion media. Transfer the tube onto a shaking device for 20 minutes at 37 degrees Celsius to digest. Make sure all pieces of tumor are submerged and not stuck to the edge of the tube.
Immediately after the digestion step, place the tube on ice to slow enzyme activity. Add EDTA to achieve a final concentration of 20 millimolar and briefly vortex the sample to mix in order to further slow enzyme activity. Open the tube and rinse any tumor digest off the lid of the tube with fresh RPMI medium. Then, prepare a 70-micron strainer. Place it on a 50-milliliter open tube on ice. Pre-wet the filter with medium. Resuspend the digested cells and wash the sides of the tube using a 25-milliliter or larger stripette. The wider opening of the stripette allows the thick digest to pass easily.
Transfer all of the digest onto the strainer using the 25-milliliter stripette. Mash up and down the tumor on top of the filter using a 1-milliliter syringe plunger. Continuously wash cells through the strainer with RPMI. Make sure to wash with enough force to push the cells through. Continue washing until all cells have passed through the strainer. Centrifuge the tube for five minutes at 300 times g and 4 degrees Celsius.
Carefully resuspend the cell pellet and complete RPMI, and pass directly through another filter to remove any extracellular matrix or large cell clumps that cannot be adequately resuspended. If no stimulation is required, immediately stain the isolated cells for flow cytometry analysis or resuspend them in freezing medium of 10% DMSO in FBS and store at minus 80 degrees Celsius.
To perform stimulation, first, count the cells. Dilute the cells in complete medium to achieve a concentration of 2 times 10 to the 6 per 100 microliters. Plate 100 microlitres of cells in each well of a U-bottomed 96 well plate. Add 100 microliters of complete medium containing a 2X preparation of PMA and ionomycin. Place the plate in an incubator at 37 degrees Celsius with 5% carbon dioxide for one hour. Then, add 20 microliters of a 10X preparation of Brefeldin-A and Monensin and complete media to achieve a final concentration of 1.06 micromolar and 2.0 micromolar, respectively. Put the plate back into the incubator for another four hours.
