The process for a one-way Mixed Lymphocyte Reaction begins by collecting whole blood from the individual whose lymphocytes will be mixed. Once the whole blood is collected, the WBC’s need to be isolated (see: Ficoll-paque separations). The isolated cells are then put into a small amount of media, and cell concentration and viability are calculated. Once these numbers are known, the cells can be put into their seeding concentration. If you are performing a one-way MLR, the stimulator cells needs to be rendered non-proliferative, so that only the proliferation of the responder cells is measured. This can be accomplished by radiation, or by treating with a DNA-crosslinker. Note that in a two-way Mixed Lymphocyte Reaction, this step is not performed.
Once all cell populations have been adjusted to their seeding concentrations, it is time to set up the plate. An equivalent number of stimulator and responder cells are placed into their proper wells, and once all the conditions are seeded, we are ready to incubate. Note, positive and negative controls are also set up at this time. For negative controls, add only media to the responder cells. For a positive control, an appropriate antigen or mitogen is added to stimulate proliferation. Once all the wells are seeded, the plate is incubated for five to seven days while the reactions occur. At the end of this time, BrdU is added, and the plates are allowed an additional 12-24 hours of incubation time, enough to allow for BrdU incorporation. Finally, the BrdU is washed out, and an ELISA is performed to determine the amount of cell proliferation. Congratulations, you can now leave the lab with your results. But you left with the question, “What does it all mean?” Let us continue on by seeing how to interpret the results of a Mixed Lymphocyte Reaction.