T Cell Purification

• Purification procedures are conducted before the experiment begins. T cells taken from the spleen or lymph nodes of animals are cleansed with a Dulbecco's Minimum Essential Medium (DMEM) wash. The wash is mixed to contain the same type of normal serum as the animal cells being assayed, penicillin-streptomycin and the chemical 2-mercaptoethanol.
  
  

Incubation

• The T cells must be incubated in an incubator at a temperature of 37 degrees Celsius for at least thirty minutes. The cells are then put into a centrifuge for a minimum of eight minutes at 800 rotations per minute.
  
  

Proliferation Assay

• T cells are dissolved using warmed DMEM and a centrifuge at 10,0000 rotations per minute. They are then transferred to a sterile container or tube and cooled in a -20 degree freezer. The T cells are proliferated by placing them into a 96 well plate and incubating in a Carbon Dioxide incubator for a few days. 0.5 to 1.0 milliCurie of three hydrogen labeled thymidine is added to the wells and incubated for an additional 15-18 hours. The cells are then put onto a glass filter for scintillation counting (radiation detection in cells).