It is widely recognized that fluorescent labeling of cells is an effective method for monitoring viable cells in a sample. Flow cytometry combined with fluorescent staining is a powerful tool to analyze heterogeneous cell populations. Among all the existing fluorescent dyes fluorescein diacetate (FDA) and its derivatives (such as CFSE) are non-fluorescent molecules that diffuse into cells and are hydrolyzed by intracellular non-specific esterases to give fluorescent products. The fluorescent products are generated and accumulated only in the cells that have intact cell membranes and active esterase activities while dead cells are not stained. The precise kinetics of membrane transport and intracellular hydrolysis of FDA and its analogs (such as CFSE) are related to cellular functions. However, it is impossible to use CFSE and its fluorescein analogs for GFP-transfected cells or for the applications where a FITC-labeled antibody is used since CFSE and its fluorescein analogs have the excitation and emission spectra almost identical to GFP or FITC. VFSE™ dyes are functionally similar to CFSE since they contain both an esterase-cleavable and an amine-reactive SE group. VFSE™ dyes can be used for the multicolor applications where either GFP or FITC-labeled antibody is used since VFSE™ dyes have either excitation or emission spectra distinct from CFSE and its fluorescein analogs.
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