Academic literature on the topic 'Activity-Based Fluorescent Probe'

Flow cytometry represents a powerful and evolving methodologic approach to cell adhesion biology. In its beginnings, flow cytometry was used solely to measure the expression of receptors on cellular surfaces and to correlate that expression with biologic function in non-flow-cytometry-based assays. From this primitive beginning, applications have proliferated and now include methodologies that measure real-time aggregation, receptor activity, and the downstream biologic consequences of cell adhesion. These biologic applications have led to platforms that are easily employed as drug screening and target validation tools. Functional assays that measure cell aggregation were initially developed to measure cell–cell interactions in the immune system, especially between cytotoxic cells and various cell types targeted as the focus of their cytotoxic activity. The cytotoxic “effector” cells and the “target” cells were stained with spectrally distinct fluorescent dyes, gently sedimented together into a cell pellet, and allowed to interact under static conditions for designated intervals of time. When resuspended and introduced into the flow cytometer, effector cells adherent to target cells were detected as “conjugate” particles emitting the fluorescence spectra of both dyes. Nonadherent effector and target cells were detected as monochromatically fluorescent particles. By using ion concentration–sensitive cytoplasmic fluorescent probes as the effector cell labels, it was also possible to detect physiological changes in intracellular ionized calcium and pH elicited by adhesion to target cells and to correlate these responses with cytotoxic function. Later, methods were developed for continuously measuring (“real-time”) cell adhesive interactions as they progressed over time in a fluid shear environment. A limitation of early adhesion kinetics analyses was that the fluid shear was generated with a magnetic stir bar and was thus neither homogeneous nor amenable to precise quantification. Subsequent refinement of these methods has enabled flow cytometric analysis of cell mixtures subjected to a more uniform and quantifiable fluid shear environment generated in a cone-plate viscometer. Cell mixtures are sampled periodically from the viscometer into a formalin fixative solution for subsequent off-line flow cytometric analysis. These experiments have been able to demonstrate a remarkable potentiation of adhesion efficiency through the combined action of two sets of adhesion molecules and a progression of adhesion molecule use from one class to another over time.