Culturing of Activated T Cells from Human Peripheral Blood Mononuclear Cells

To begin, wash CD3/CD28 beads by transferring 12.5 microliters of beads per million cells to a microcentrifuge tube, and adding 12.5 microliters of PBS per 12.5 microliters of the beads in the tube. Then, place the microcentrifuge tube on a suitable magnet for 1 minute, discard the buffer, and resuspend the beads in the original volume of T-cell medium.

Next, add 12.5 microliters of beads per million cells at a 1 to 2 beads to cells ratio, and divide the cells into two conditions with around 5 million cells in each. Then, add the correct volume of cytokines to each condition, and transfer the cells to multi-well plates. Incubate the cells for three days at 37 degrees Celsius and 5% carbon dioxide.

After three days of incubation, prepare fresh T-cell medium with double the concentration of cytokines. Resuspend and split the cells by transferring half the volume from each well into a new well. Then, add the same volume of freshly-prepared T-cell medium to each well.