4.4. T Cell Proliferation Assay

Draining lymph node (DLN) cells and splenocytes were isolated from EAE control and analogue-treated rats 10 days after immunization with MBP_72–85 and isolated cells were cultured for 72 h in 96-well plates in RPMI 1640 containing 10% heat-inactivated FCS, 50 μM 2-mercaptoethanol (all Invitrogen Life Technologies, Gaithersburg, MD, USA) and increasing concentrations of MBP_72–85 agonist or cyclo(87–99)[Arg⁹¹, Ala⁹⁶]MBP_87–99 analogue. Cells were pulsed with 1 μCi/5 × 10⁵ cells [3H]thymidine (PerkinElmer, Waltham, MA, USA) for the last 16 h of culture, and [3H]thymidine incorporation was measured by liquid scintillation counting (Wallac, Turku, Finland). Results are expressed as stimulation index (ratio between radioactivity counts of cells cultured in the presence of Ag and of cells cultured with medium alone).