PSLs affect splenic cognate antigen specific proliferation and expression of TNFα, iNOS and ARG-1. (a) Cognate antigen specific proliferation (10 dpi) of splenic cultures was assessed by culturing splenic cells from vehicle, PCL and PSL treated animals with MOG. Proliferation was assessed by [3H]thymidine incorporation. Non-stimulated cultures were used as control (dotted line). Data represent the mean ± SEM of four experiments. (b) The size of the splenic white pulp was determined using ImageJ software. Three cryosections per animal were stained with CD200R, a marker for myeloid cells, after which the surface area surrounded by the marginal metallophilic macrophages was determined. Five images (4× magnification) per section were taken to calculate the mean white pulp size. Data represent the mean ± SEM of four animals. (c,d) Comparison of fold changes between vehicle, PCL and PSL-treated spleens 10 dpi. Relative quantification of iNOS (c) and TNFα (d) was accomplished by using the comparative Ct method. Data were normalized to the most stable reference genes, determined by Genorm (Pgk1 and Rpl13a). Data represent the mean ± SEM of 4 experiments. (e) Spleen cryosections were stained with ARG-1 after which the total corrected fluorescence was determined using ImageJ software, as described previously . Three cryosections were stained and 6 images (10× magnification) were taken per section. Data represent the mean ± SEM of four animals.