Data collected from dead cells often give false positive results as they tend to bind nonspecifically to many fluorescent reagents used in experiments. Therefore, removing dead cells from your flow cytometry data is a critical step to insure accurate results and analysis. Life Technologies provides a unique set of violet laser-compatible Molecular Probes® fluorescent dyes designed to easily and accurately define live and dead cell populations.
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The fixable dead cell stains covalently interact with available surface amines, and are excluded from the interior of healthy cells. In contrast, for dead cells, the dyes label proteins throughout the cytoplasm, staining dead cells with at least 50-fold greater fluorescence than live cells. Because the labeling is covalent, stained cells can be aldehyde-fixed and permeabilized without losing viability discrimination, making it ideal for researchers who want to fix samples before analysis. The ArC™ Amine Reactive Bead Kit is the perfect tool for setting up the compensation requirements of the fixable dead cell stains in your experiments.
- Postfixation measurement of viability
- Accurate intracellular staining measurements
- Reliable viability measurements
Use of fixable dead cell dyes. The reagents in the LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit were used to differentially stain a mixture of live (left peak) and heat-treated Jurkat cells (right peak). The cells in (A) were not fixed; the cells in (B) were fixed in 3.7% formaldehyde following the staining reaction. Samples were analyzed by flow cytometry using 405 nm excitation and ~525 nm emission.
SYTOX® Blue Dead Cell Stain is a high-affinity nucleic acid stain that easily penetrates cells with compromised plasma membranes but remains excluded from healthy cells. After brief incubation with SYTOX® Blue Dead Cell Stain, the nucleic acids of dead cells fluoresce bright blue when excited with 405 nm violet laser light. These properties make the SYTOX® Blue Dead Cell Stain a simple and quantitative single-step dead cell indicator for use with violet laser–equipped flow cytometers.
- Alternative laser excitation for nucleic acid staining
- Replacement for propidium iodide (PI) using the violet laser
- Rapid and easy-to-use protocol for DNA staining
Use of SYTOX® Blue Dead Cell Stain with Vybrant® DyeCycle™ Green Stain. Jurkat cells were stained with Vybrant® DyeCycle™ Green Stain, then stained with the impermeant DNA dye, SYTOX® Blue Dead Cell Stain, and analyzed by flow cytometry using 405 nm and 488 nm excitation, gating on SYTOX® Blue–negative cells to eliminate dead cells. Cell cycle was then evaluated using a histogram of Vybrant® DyeCycle™ Green staining.
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CellTrace™ Calcein Violet AM is an optimal dye for detecting intracellular esterase activity using the violet laser. It is available either as a stand-alone reagent or in the LIVE/DEAD® Violet Viability/Vitality Kit, which combines CellTrace™ Calcein Violet fluorescence with the dead cell aqua–fluorescent reactive dye. This unique kit allows the simultaneous analysis of both live and dead cells using the violet laser.
- Live cell stain for the violet laser
- Determines physiological activity regardless of membrane status
- Little to no non-specific background staining
Combination of esterase substrates with dead cell dyes using mixtures of heat-treated and untreated cells. Chinese hamster ovary (CHO) cells were stained according to the protocol in the LIVE/DEAD® Violet Viability/Vitality Kit (CellTrace™ Calcein Violet , AM and fixable violet dead cell stain). Cells were analyzed by flow cytometry using 405 nm excitation.
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Determination of live and dead cells and absolute cell number
Plot of calcein fluorescence collected with 530/30 nm bandpass filter vs. ethidium homodimer-1 fluorescence collected with 610/20 nm bandpass filter, showing clear separation of live and dead cells, as well as counting beads (C36950). A mixture of live and heat-killed Jurkat cells (human T-cell leukemia) was treated with the reagents in the LIVE/DEAD® Viability /Cytotoxicity Kit, counting beads were added, and the sample was analyzed by flow cytometry using 488 nm excitation.
|CountBright™ Absolute Counting Beads||product details|