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Ex vivo splenocyte culture

 
  1. Remove spleen and grind up using the outer end of a 10cc syringe.
  2. Spin down at 1000 rpm for 10 min and resuspend in 5 ml ACK lysis buffer.  Lyse for 2 min, add 5 ml medium and spin down.  Wash 2x with medium, filter to remove chunks.  Count cells and resuspend at 2-4x106 cells/ml. (Usually get about 108 cells/spleen)
  3. Add 1 ml of splenocytes per well of a 24 well plate.  Combine with 1 ml medium containing 20 units/ml IL-2 (4000 units/ml stock) and either irradiated splenocytes or irradiated CT26-B7 (see T cell clone protocol).  If using irradiated splenocytes add 20 μg peptide per well (2 μl of a 10 mg/ml stock). 
  4. Re-stimulate cells once a week.