A Technique for the Generation of Antigen-Specific CAR T Cells
Trascrizione
On day 1, prepare human fibronectin fragment-coated plates by adding 0.5 milliliters of Fibronectin to the wells of a 24-well plate and incubating it overnight at 4 degrees Celsius.
On the next day, remove the fibronectin solution and add 1 milliliter of 2% BSA in PBS per well. Incubate the plate at room temperature for 30 minutes, and wash the treated wells with 1 milliliter of sterile PBS. The plate is ready to use after removing wash solution.
Count the activated CD8 T cells using trypan blue. Collect them by centrifugation, and resuspend them at 5 million viable cells per milliliter for transduction. Add 100 microliters of the activated CD8 cell suspension to each well of the fibronectin-coated plate. Then, add 1.5 to 2 milliliters of the previously prepared virus-containing medium, and mix using a swirling motion to evenly dispense the cells.
Seal the plate in a zip-lock bag and centrifuge it at 2000 times g for 90 minutes at 37 degrees Celsius. Remove the plate from the centrifuge and take it to a biological safety cabinet. Carefully remove the plastic bag and ensure that the outside of the plate is not contaminated with medium. Transfer the plate to a 37 degrees Celsius carbon dioxide incubator.
After 4 hours, remove 1 milliliter of medium from each well. Replace it with 1 milliliter of pre-warmed complete T cell medium and put the plate back in the incubator.
