Bene L, Fulwyler M J, Damjanovich S
Department of Biophysics and Cell Biology, University Medical School, University of Debrecen, Hungary.
Cytometry. 2000 Aug 1;40(4):292-306.
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.
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.
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.
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.
佩兰方程提出了一种通过在传统流式细胞仪中测量极化供体强度，在逐个细胞基础上准确测定能量转移的替代方法。
通过流式细胞术生成活细胞表面荧光抗体标记的MHC I类和II类分子的佩兰寿命图，研究供体的能量转移与荧光各向异性之间的关系。能量转移降低了供体的荧光寿命。
佩兰图已被证明对标记染料和不同同种型抗体的片段迁移率以及由于抗体多重标记导致的同源转移敏感。提出了一种通过测量供体各向异性增强来确定能量转移可行性的方法。展示了供体各向异性和推导的能量转移效率的流式细胞术直方图，表明MHC I类和II类分子在人T淋巴母细胞表面聚集。在附录中，还提出了一种无需测量G因子即可同时测定能量转移效率和供体荧光各向异性的方法。
我们证明了在流式细胞仪中可以同时测量适当选择的供体和受体之间的能量转移效率，即接近度，以及供体的旋转弛豫，即供体迁移率。
