Fluorescence Polarization as an Early Measure of T-Lymphocyte Stimulation

Transmembrane stimulation of lymphocytes at the G₀ –G₁ resting phase, induced by specific antigens, mitogens, or by antibodies to certain cell surface molecules, results in a complex series of well-characterized molecular events, culminating in lymphocyte activation, transformation, mitosis, and finally apoptosis (1 –3 ). These events are associated with early changes in membrane potential, coupled with an Na⁺ influx, and with changes in pH, followed by the influx and internal release of Ca²⁺ ions. The processes linking early and late intracellular events in the course of cell activation involve conformational changes of cytosolic enzymes and/or their regulatory proteins, as well as their intracellular matrix reorganization (4 –6 ). The monitoring of these early structural changes can be performed by measuring the fluorescence polarization (FP) of intracellular fluorescent probes (7 –13 ). Specific fluorescence probes such as diphenyl hexatriene (DPH) and fluorescein diacetate (FDA) have been used to measure membrane fluidity (14 ,15 ) and changes in the cytoplasmic microviscosity (7 ), respectively.