Detection of receptor clustering by flow cytometric fluorescence anisotropy measurements

Background: Perrin equation suggests an alternative way for the accurate en ergy 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 anisotro py of the donor was investigated by flow cytometric generation of Perrin-li fetime plots of fluorescent antibody-labeled MHC class I and class Il molec ules on the surface of living cells. The energy transfer reduced the fluore scence lifetime of the donor. Results: Perrin plots ha ve proven to be sensitive to the segmental mobilit y of the labeling dye and that of antibodies of different isotypes, and hom e-transfer due to the multiple labeling of antibodies. A method demonstrati ng the feasibility of energy transfer determination by measuring anisotropy enhancement of the donor is presented. Flow cytometric histograms of the d onor anisotropy and of the deduced energy transfer efficiency are shown, in dicating clustering of MHC class I and class II molecules on the surface of human T lymphoblasts. In the Appendix, a method for the simultaneous deter mination of both energy transfer efficiency Mel donor fluorescence anisotro py, without need for G-factor measurement, is also presented. Conclusions: We demonstrate that energy transfer efficiency, i.e., proximit y, between suitably selected donor and acceptor, and the rotational relaxat ion of the donor, i.e., donor mobility, can be simultaneously measured in a flow-cytometer. (C) 2000 Wiley Liss, Inc.