Detection of receptor clustering by flow cytometric fluorescence anisotropy measurements.
BACKGROUND
Perrin equation suggests an alternative way for the accurate energy transfer determination on a cell-by-cell basis by measuring polarized donor intensities in a conventional flow cytometer.


METHODS
The relationship between energy transfer and fluorescence anisotropy of the donor was investigated by flow cytometric generation of Perrin-lifetime plots of fluorescent antibody-labeled MHC class I and class II molecules on the surface of living cells. The energy transfer reduced the fluorescence lifetime of the donor.


RESULTS
Perrin plots have proven to be sensitive to the segmental mobility of the labeling dye and that of antibodies of different isotypes, and homo-transfer due to the multiple labeling of antibodies. A method demonstrating the feasibility of energy transfer determination by measuring anisotropy enhancement of the donor is presented. Flow cytometric histograms of the donor anisotropy and of the deduced energy transfer efficiency are shown, indicating clustering of MHC class I and class II molecules on the surface of human T lymphoblasts. In the Appendix, a method for the simultaneous determination of both energy transfer efficiency and donor fluorescence anisotropy, without need for G-factor measurement, is also presented.


CONCLUSIONS
We demonstrate that energy transfer efficiency, i.e., proximity, between suitably selected donor and acceptor, and the rotational relaxation of the donor, i.e., donor mobility, can be simultaneously measured in a flow cytometer.
Department of Biophysics and Cell Biology, University Medical School, University of Debrecen, Hungary.
L
L
Bene
M J
MJ
Fulwyler
S
S
Damjanovich
D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology; D002478:Cells, Cultured; D004735:Energy Transfer; D005434:Flow Cytometry / Q000379:methods*; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D000949:Histocompatibility Antigens Class II / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D011956:Receptors, Cell Surface / Q000032:analysis*; D013601:T-Lymphocytes / Q000276:immunology; D001613:beta 2-Microglobulin / Q000276:immunology
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D007074:Immunoglobulin G; D011956:Receptors, Cell Surface; D001613:beta 2-Microglobulin