IN-VIVO LABELING OF THE SPLEEN WITH A RED-FLUORESCENT CELL DYE

A method for labelling mouse spleen cells in situ is described. Spleen vessels were clamped before intrasplenic injection of a red-fluoresce nt cell dye (PKH-26). The labelling rate was 11.8% of all cells and 13 .9% of B lymphocytes 30 min after injection as determined by FACS. 3 d ays later, the proportions of labelled cells were reduced to 7.4% (P < 0.01) and to 10.7% (P < 0.05), respectively. Only 10% of cells detect ed by FACS could be detected by fluorescence microscopy. Labelled cell s were not found in peripheral lymph nodes 30 min after spleen injecti on, but were present 3 days later (FACS: 2.8% of all cells and 5.4% of B lymphocytes, P < 0.05 each), indicating migration of stained cells. Neither adverse nor toxic effects of in situ staining were observed. Isolated stained B lymphocytes from spleens of naive mice and from lym ph nodes after immunisation with insulin showed proper function when t ested in an ELISA-spot assay. The labelling procedure was used to foll ow splenic B lymphocytes producing natural anti-insulin antibody. Thes e cells were found to be recruited for the early immune response in ly mph nodes immunised with insulin.