Repeated stimulation assay

Experiment set-up (2:1 Effector to Target (E:T) ratio):

1. Harvest TARGET (tumor) cells; resuspend in tumor cell media at 5 x105 cells/ml and plate 1mL (5 x105 cells) per well of 24 well plate; add 1ml media to control well. Let them attach (1hr or so)

2. Harvest CAR T cells (T cells within 5-12 day after transduction)

3. Spin 400xg for 5 minutes, resuspend at 1 x106 cells/cc in T cell media and plate 1mL (1 x106 cells) per well of 24 well plate. Add IL-15 to the co-culture (5ng/mL).

4. After 24 hours remove 500 ul of media (snap freeze); add 500 ul fresh media (for ELISA or Miliplex)

5. Day 7:

   a. re-stimulate cells: count T cells/well* and re-plate them on fresh tumor cells at 2:1 E:T ratio (steps 1-3).

*- count cells using hemocytometer (total cell number: cell/ml x total volume)