The violet laser (405 nm) has become increrviceseasingly popular in flow cytometry due to its ability to increase the multiplexing capabilities of the flow cytometer. Life Technologies is the leader in providing reagents for the violet laser for the analysis of cellular processes including cell cycle analysis, cell viability and vitality, cell proliferation, and apoptosis. These reagents can be used on the violet laser channels to increase the multicolor capabilities of today’s flow cytometers. Using the violet laser for these parameters frees the other channels on the flow cytometer for immunophenotyping and other common flow applications.
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Cell Cycle Analysis
Vybrant® DyeCycle™ Violet stain is a DNA-selective stain for use in live cells. Cell cycle analysis using this stain results in a clear pattern of distribution: G0/G1 phase (one set of paired chromosomes per cell), S phase (DNA synthesis with variable amount of DNA), and G2/M phase (two sets of paired chromosomes per cell, prior to cell division). This live-cell dye allows the simultaneous co-staining of live cells for other parameters and offers the possibility of cell sorting based on DNA content and identification of stem cell side populations.
- Determines cell cycle status of living cells
- Able to identify stem cell side populations
- Limited cytotoxicity
|Cell cycle analysis using Vybrant® DyeCycle™ Violet Stain. Histogram of live Jurkat cells stained with Vybrant® DyeCycle™ Violet Stain showing DNA content distribution. G0/G1- and G2/Mphase histogram peaks are separated by the S-phase distribution. Violet 405 nm excitation was used with a 440/40 nm bandpass filter.
|Vybrant® DyeCycle™ Violet Stain||product details|
FxCycle™ Violet Stain is a violet laser–excited dye used for cell cycle analysis in fixed cells. Using FxCycle™ Violet for cell cycle analysis increases the ability to multiplex by freeing up the 488 nm and 633 nm lasers for other cellular analyses such as immunophenotyping, apoptosis analysis, and dead cell discrimination. Because of its narrow emission spectra, FxCycle™ Violet Stain overlaps less with other fluorescent channels than other commonly used dyes (propidium iodide and 7-AAD), resulting in minimal compensation requirements and more accurate data.
- Flexibility for multicolor cell cycle studies
- Limited compensation requirements for 488 nm excitable dyes
- Tight coefficient of variation
Multiparametric cell cycle and immunophenotypic analysis. TF-1 erythroblast cells were alcohol-fixed overnight, washed, and then suspended in 0.1% Triton® X-100/PBS/1% BSA before staining with anti–histone H3[pS10] purified antibody complexed with Zenon® Alexa Fluor® 488 Rabbit IgG labeling reagent and FxCycle™ Violet stain. The pH3 signal (red) identifies cells that are in mitosis.
|FxCycle™ Violet Stain||product details|
Cell Viability and Vitality
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.
|SYTOX® Blue Dead Cell Stain||product details|
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.
|LIVE/DEAD® Violet Viability/Vitality Kit||product details|
|CellTrace™ Calcein Violet, AM||product details|
Unlike the traditional method of BrdU incorporation of analyzing new DNA synthesis, Click-iT® EdU Pacific Blue™ Flow Cytometry Assay Kit eliminates the need to denature DNA in order to fluorescently label the incorporated nucleoside. The result is a much simplified, consistent method of analyzing cell proliferation by flow cytometry with the further advantage of being able to include additional parameters in your experimental protocol.
- Accurate, consistent performance—no denaturation steps or harsh treatments required
- Simple method—works the first time, every time, in less time
- Content-rich results—better preservation of cell morphology, antigens, and dsDNA integrity
Flow cytometric analysis of cell proliferation using Click-iT™ technology. Jurkat cells were treated with 10 μM EdU for 2 hr, then fixed and permeabilized, stained with the click reaction, washed, and counterstained for cell cycle analysis using 7-aminoactinomycin D (7-AAD). Cells were analyzed using a flow cytometer with 405 nm excitation and 450⁄50 nm bandpass and 675⁄20 nm bandpass filters.
|Click-iT™ EdU Pacific Blue™ Flow Cytometry Assay Kit||product details|
Apoptosis is a carefully regulated process of cell death that occurs as a normal part of development. It is distinguished from necrosis, or accidental cell death, by characteristic morphological and biochemical changes. Two Vybrant® Apoptosis Assay Kits incorporate a violet-excitable stain as part of a two-color assay for the detection of apoptosis. The Pacific Blue™–annexin conjugate detects changes in membrane asymmetry characteristic of apoptotic cells. In addition to changes in membrane asymmetry, slight changes in permeability to dyes such as PO-PRO™-1 (also available in Vybrant® Apoptosis Assay Kit #13 PO-PRO™-1/7-aminoactinomycin D) can be used to identify apoptotic cells. Unlike annexin conjugates, PO-PRO™-1 can provide efficient staining of both trypsinized and suspension cells.
- Detection of multiple apoptotic parameters on the violet laser
- Increased multiplexing capabilities
- Available as stand-alone reagents or easy-to-use kits
Violet-excited DNA stain for apoptosis detection. Jurkat cells were treated with 10 μM camptothecin for 4 hr and then stained with the reagents in the Vybrant® Apoptosis Assay Kit #13 PO-PRO™-1/7-aminoactinomycin D. Cells were analyzed by flow cytometry using 405 nm and 488 nm excitation. Apoptotic cells (A) are clearly distinguished from live cells (L) and dead cells (D).
|Annexin V, Pacific Blue™ Conjugate||product details|
|Vybrant® Apoptosis Assay Kit #13 PO-PRO™-1/7-aminoactinomycin D||product details|
|Vybrant® Apoptosis Assay Kit #14 Annexin V-Pacific Blue™||product details|
CountBright™ Absolute Counting Beads
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|