A method has been developed for the determination of the efficiency (E) of the fluorescence resonance energy transfer between moieties on cell surfaces by use of a computer-controlled flow cytometer capable of dual wavelength excitation. The absolute value of E may be calculated on a single-cell basis. The analysis requires the measurement of samples stained with donor and acceptor conjugated ligands alone as well as together. In model experiments HK 22 murine lymphoma cells labeled with fluorescein-conjugated concanavalin A (Con A) and/or rhodamine conjugated Con A were used to determine energy transfer histograms. Using the analytic solution to energy transfer in two dimensions, a high surface density of Con A binding sites was found that suggests that the Con A receptor sites on the cell surface are to a degree preclustered . We call this technique flow cytometric energy transfer ( FCET ).

Change of osmolality surrounding spawned sperm from isotonic to hypotonic causes the initiation of sperm motility in the common carp. Here we show that membrane-permeable cAMP does not initiate motility of carp sperm that is quiescent in isotonic solution, and that motility of the demembranated sperm can be reactivated without cAMP. Furthermore, the cAMP level does not change during the initiation of sperm motility, and inhibitors of protein kinase do not affect sperm motility, suggesting that no cAMPdependent system is necessary for the regulation of sperm motility. Sperm motility could not be initiated in Ca 2؉ -free hypoosmotic solutions, and significant increase in the intracellular Ca 2؉ level was observed by a Ca-sensitive fluorescence dye during hypoosmolality-induced active motion period. The demembranated sperm cells were fully reactivated in the solutions containing 10 ؊7 to 10 ؊5 M Ca 2؉ . Ca 2؉ channel blockers such as verapamil and -conotoxin reversibly inhibited the initiation of sperm motility, suggesting that Ca 2؉ influx is the prerequisite for the initiation of carp sperm motility. Motility of intact sperm was completely blocked; however, that of the demembranated sperm was not inhibited by the calmodulin inhibitor W7, suggesting that the calmodulin bound close to the plasma membrane participated in the initiation of sperm motility. Flow cytometric membrane potential measurements and spectrophotometric measurements by using fluorescence dyes showed transient membrane hyperpolarization on hypoosmolality-induced motility. This article discusses the role of membrane hyperpolarization on removal of inactivation of Ca 2؉ channels, leading to Ca 2؉ influx at the initiation of carp sperm motility.

The energy transfer efficiency E was measured between fluorescein-conjugated concanavalin A (Con A) and rhodamine-conjugated Con A bound to homogeneous tissue culture cells, the HK22 murine lymphoma cell line. Results from a flow cytometric energy transfer method (FCET) and two different steady-state fluorimeter methods were compared. The data were found to be in close agreement after careful correction of the steady-state fluorimetric measurements for contributions from dissociating ligand. The biological variability of the individual cells with respect to E was calculated using an error propagation analysis and were found to be less than the variability in the absolute amount of ligand binding per cell. FCET has a number of advantages over the fluorimetric measurements using suspensions of cells: (1) relatively labile receptor-ligand complexes can be measured; (2) the analysis can be restricted to undamaged cells by gating the data collection on the light-scattering signals; (3) heterogeneous populations of cells with respect to donor and acceptor topology can be distinguished by the correlation of E with other cellular parameters derived from additional signals or combinations thereof; and (4) the dynamics of donor-acceptor redistribution on subpopulations can be measured.Key terms: Lectins, receptor, flow analysis, flow cytometryThe state of receptor aggregation in the cytoplasmic membrane may play a central role in the mediation between external stimuli and the complex metabolic processes of the cell. For example, binding of epidermal growth factor (111, nerve growth factor (91, cr2-macroglobulin (lo), and insulin (12) to their corresponding receptors results in a redistribution of the latter followed by a series of prompt biological responses, and, in the case of the hormones, initiation of DNA synthesis and stimulation of growth. The importance of receptor redistribution for normal metabolism has been investigated with mutant cells. Anderson et a1 (1) demonstrated that in one genetic form of the metabolic disorder hypercholesterolemia the low-density-lipoprotein receptors can no longer cluster, a situation that prevents the internalisation of the protein receptor complex. Specific cell types or different functional surfaces of differentiated cells may exhibit distinct spatial distributions of surface proteins, eg, as in the case of Con A binding glycoproteins on endothelial cells (13). The role of such microdomains in cell-cell recognition or functional compartmentalization is the subject of many current investigations.One of the most useful techniques for studying proximity relationships (intermolecular distances) in the range that pertains to the cell membrane is fluorescence resonance energy transfer. The method has been applied to the study of cell surface phenomena using steadystate fluorimeters (51, fluorescence microscopes (6), and flow cytometers (4,18,14). We have recently developed a method for the quantitative evaluation of the energy transfer efficiency (E) between donors and acceptors on cell...

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