In vivo labelling 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-fluorescent 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 days 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 detected by FACS could be detected by fluorescence microscopy. Labelled cells were not found in peripheral lymph nodes 30 min after spleen injection, 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 lymph nodes after immunisation with insulin showed proper function when tested in an ELISA-spot assay. The labelling procedure was used to follow splenic B lymphocytes producing natural anti-insulin antibody. These cells were found to be recruited for the early immune response in lymph nodes immunised with insulin.
Khalaf
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AN
Abteilung Klinische Endokrinologie, Medizinische Universitätsklinik, Freiburg, Germany.
Wolff-Vorbeck
G
G
Bross
K
K
Kerp
L
L
Petersen
K G
KG
D000818:Animals; D001402:B-Lymphocytes; D002465:Cell Movement; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D008198:Lymph Nodes / Q000166:cytology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D009930:Organic Chemicals; D013154:Spleen / Q000166:cytology*
D005456:Fluorescent Dyes; D009930:Organic Chemicals; C070080:PKH 26