Flow Cytometry - Applications

Fixable Viable Dyes

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The advent of increasingly complex polychromatic flow cytometric applications combined with the phenotypic identification and quantification of rare cell subsets, neccesitates the addition of a viable dye in the multicolor mix. It is a well known fact that dead and /or dying cells undergo biochemical changes that render their membranes "sticky" and increase their level of autofluorescence relative to viable cells. These non-viable cells can non-specifically bind to antibodies, thereby resulting in high levels of background and false-positive binding that can significantly impact phenotypic characterization and/or quantitation of rare cells within a heterogeneous population. Previously, popular methods for excluding non-viable cells included the use of impermeant "viability" dyes that stain necrotic or apoptotic cells based on the ability of these dyes (generally DNA-binding dyes) to penetrate only non-intact membranes and then intercalate into DNA base-pairs and fluoresce. These membrane impermeant dyes could not be used on fixed cells, as fixation facilitates dye entry into the entire population. An alternative dye called EMA (Ethidium Mono-Azide) was often employed in the past. This dye also binds to DNA, however, it can be crosslinked via a short exposure to UV light, thereby rendering the cells fixable, without loss of the dye, and preserving viable/non-viable delineation. EMA, once bound and crosslinked to DNA flouresces in the same emission range as FITC, thereby eliminating that channel for use in multicolor analysis.

A recent article in JIM describes a commercially available class of amine-reactive dyes introduced by Invitrogen/Molecular Probes that overcome the technical issues typically associated with traditional viable dyes, and allows for fixation and/or subsequent permeabilization of cells following reaction with the dye. Thus viability determination is possible even when extensive manipulation to the cell membranes is required for detection of intracellular epitopes. A further benefit of these dyes is their availability in a multitude of colors, including blue, green, red, violet, and aqua, to allow flexiblity in designing multicolor antibody panels. We have done testing on several colors in the lab, including red, violet, and aqua, and have found the dyes simple to use and extremely effective and reproducible in defining viable populations. We particularly like the violet and aqua dyes as they are excited by the violet laser and have little spectral overlap with commonly used fluorochromes. These dyes are av ailable as "LIVE/DEAD Fixable Dead Cell Stain Kits".

Journal of Immunological Methods 313: 199-208, 2006.

Apoptosis Detection in Adherent Cells: FLICA Reagents

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An alternative assay that may prove more reproducible and circumvent the above issue in adherent cells employs a novel methodology to detect active caspases based on a fluorescent inhibitor of caspases (FLICA) reagent. This reagent is comprised of a fluoromethyl ketone (FMK) moiety associated with a conserved caspase recognition sequence in peptide form, attached to a carboxyfluorescein group (FAM) as a reporter (525nm emission). This cell-permeant, non-cytotoxic FLICA reagent is believed to interact with the reactive site of the activated caspase via the recognition sequence, and then to attach covalently through the FMK moiety. The unbound FLICA reagent is then washed away, providing a direct measure of the amount of active caspase at the time the inhibitor was added. This reagent can be used in combination with a viable dye, additional fluorochromes for multicolor studies, as well as fixatives, to preserve the signal for analysis at a later date. On a cautionary note, appropriate controls for back ground binding in untreated cells must be included in the assay, as FLICA substrates have been found to bind non-specifically to intracellular sites lacking caspase activity. In our hands, the data generated with the FLICA reagent replicated results obtained using western blot detection of cleaved caspases in adherent cells treated with increasing dosages of a chemotherapeutic agent, whereas the Annexin V assay did not.

Exp Cell Res 259: 308-313, 2000.
Cytometry 44: 73-82, 2001.