To begin, place a vial of cryopreserved human peripheral blood mononuclear cells in a water bath at 37 degrees Celsius. Once thawed, transfer the PBMC suspension into a 15 milliliter tube containing nine milliliters of TGM. Centrifuge the tube at 300 G for five minutes after pipetting out an aliquot of the cell suspension for counting.
Resuspend the pelleted PBMC in three milliliters of TGM. Then, transfer the required volume of cell suspension into a six well plate. The day before the cell resuscitation, transfer 100 microliters of mouse immunoglobulin G magnetic beads into a 1.5 milliliter micro centrifuge tube.
Add one milliliter PBS to the tubes and place them on a magnetic stand to wash the beads. Resuspend the beads in 100 microliters of PBS. Then add the antibodies to the suspension.
Use a pipette to gently mix the suspension well. Place the suspension on a rocker at four degrees Celsius overnight. After washing the beads in PBS as done earlier, resuspend them in 100 microliters of PBS.
Now add NHCD3 hCD28 coated immuno beads to the plate and incubate. After 48 hours of incubation, mix and transfer the cell suspension into a 15 milliliter centrifuge tube. Place the tube on a magnetic stand for three minutes.
Then transfer the supernatant into a new 15 milliliter tube. Seed five times 10 to the power of five PBMC in 300 microliters of TGM into the wells of a 48 well flat plate. Pipette 200 microliters of lentiviral stock into the corresponding wells.
Then add protamine sulfate to a final concentration of 10 micrograms per milliliter before centrifuging. Pipette out 300 microliters of the supernatant from each well, then add one milliliter of fresh TGM into each well. Place the plate in a humidified incubator at 37 degrees Celsius under 5%carbon dioxide.
Add fresh 0.5 to two milliliters TGM to the plate every two to three days. Then transfer the cells into a 12 well plate, followed by a six well plate until the total cell density reaches 6 million in a four milliliter volume.
