To begin, plate the cultured CAR-transduced T cells into two 12-well plates containing two milliliters of TGM. Place one plate directly in a humidified incubator under 5%carbon dioxide at 37 degrees Celsius. Place the other plate in a mobile carbon dioxide, oxygen nitrogen incubator chamber with the oxygen level preset at 1%Collect 500, 000 cells from the plates every 24 hours under both conditions into 1.5 milliliter micro centrifuge tubes.
Centrifuge the tubes at 500 G for five minutes. After removing the supernatant, gently pipette one milliliter of PBS into the cell pellet. Resuspend the pelleted cells in 50 microliters of FACS buffer.
Then add 50 microliters of a one to 100 dilution of phycoerythrin conjugated anti-flag antibody. Pipette the suspension to mix thoroughly. After incubation, add one milliliter of FACS buffer to each tube.
Centrifuge the mixed suspension at 500 G for five minutes. Now, pipette out the supernatants from each tube. Then resuspend the cells in 200 microliters of FACS buffer.
Transfer the resulting cell suspension into five milliliter flow cytometry tubes. Perform flow cytometry on the cell suspension to determine the surface chimeric antigen receptor expression. Set up new tubes as blank and CAR.
Click FSCA, FSCH, SSCA, SSCH, PE and FITC channel. Then create four scatter plots to sequentially identify single cells, living cells, GFP positive and PE positive cells. Set parameters for collecting 10, 000 single living cells.
Click acquire data. After cell collection is stable, click record data. To determine the location of gates according to negative control, gate the cells positive for EGFP and phycoerythrin to measure phycoerythrin positivity and median fluorescence intensity.
The hypoxia sensitive per two targeting CAR showed significantly high expressions of CAR under hypoxic conditions relative to normoxic conditions.
