Quantifizierung von Histokompatibilitätsantigenen auf Rattenlymphozyten mittels Durchflußzytometrie

Summary

For studies on quantitative aspects of MHC-coded surface antigens on rat lymphocytes, cell separation methods were combined with flow cytometry, using direct immunofluorescence and titration to antibody saturation. Lymphocyte subpopulations were isolated from spleen, lymph nodes, thymus and bone marrow of BN rats by 1 × g sedimentation and free-flow electrophoresis. They were incubated with fluorescein conjugated F(ab')₂ antibody fragments directed against MHC-antigens. Flow cytometry showed fluorescence intensities clearly separated from background signals, increasing with antibody concentration. Mean fluorescence intensities of the cells were compared at plateau level of antibody uptake and revealed substantial differences in the quantitative expression of these antigens:

• a) B cells bound larger amounts of the antibody than T cells.

• b) B cells from spleen and lymph nodes differed in antibody uptake.

• c) In the T lineage medullary thymocytes showed higher antigen expression than cortical thymocytes, but still lower than mature T cells.

• d) The dominant, Thy-1 antigen carrying lymphocyte population of bone marrow showed a density of MHC-antigens between that of thymocytes and mature peripheral lymphocytes.

Calibration with particles of known FITC content led to an estimation on the total amount of histocompatibility antigens expressed on lymphocytes, which seems to be a characteristic feature of certain stages in function, maturation and activation.