To begin using a serological pipette, remove the growth media from the T75 flask containing 70%confluent MDA-MB-231 culture. Add three milliliters of trypsin to the flask and incubate at 37 degrees with 5%carbon dioxide for three to five minutes to detach the cells from the flask. Then to neutralize the trypsin, add three milliliters of DMEM into the flask.
Collect the cell suspension in a centrifuge tube and spin at 400 G for four minutes. Discard the supernatant using a pipette and resuspend the pellet in two milliliters of PBS. Using a cell counter, determine the cell concentration.
Transfer eight times 10 to the fifth cells into a 15 milliliter tube and add PBS to make the volume to one milliliter. Then add two microliters of CFSE into the tube and mix the cell suspension thoroughly using a one milliliter pipette. Place the tube at 37 degrees Celsius and 5%carbon dioxide incubator for 20 minutes.
Next, add five milliliters of DMEM to the tube and centrifuge to pellet down the CFSE labeled cells. Discard the supernatant and resuspend the pellet in one milliliter of DMEM. After reevaluating the cell concentration, transfer four times 10 to the fifth cells into a 25 milliliter reagent reservoir.
Add DMEM to make the volume eight milliliters and mix the cell suspension with a five milliliter serological pipette. Using a multichannel pipette, transfer 100 microliters of cell suspension into each row on the left half of a black 96-well plate. Similarly, add EGFR knockout cell suspension on the right half of the plate.
To ensure uniform cell distribution, move the plate back and forth and side to side on the platform. Incubate the plate for four hours at 37 degrees Celsius and 5%carbon dioxide for the tumor cells to attach. Based on counts of untransduced and CAR-expressing Jurkat cells, transfer four times 10 to the fifth CAR-J cells into a 25 milliliter reservoir.
Add DMEM to the reservoir to a final volume of two milliliters. For an effector to tumor ratio of 4:1, add two times 10 to the fourth CAR-J cells per well in 100 microliters of medium along the side of the well without disturbing the attached tumor cells. Then add 100 microliters of DMEM to the side of the wells containing tumor and Jurkat cells to get 300 microliters of volume per well.
Move the plate as demonstrated to ensure uniform distribution of the Jurkat cells on the tumor cells. Allow the co-culture to establish at 37 degrees Celsius with 5%carbon dioxide for 48 hours.
