Violet Laser Reagents for Cell Cycle Analysis

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. Invitrogen 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

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

Vybrant DyeCycle
 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.


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Vybrant® DyeCycle™ Violet Stain Order Now
FxCycle™ Violet Stain

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
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.

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FxCycle™ Violet Stain


Cell Proliferation

Click-iT® EdU Pacific Blue™ Flow Cytometry Kit
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
Click-iT™ Technology
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.

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Click-iT™ EdU Pacific Blue™ Flow Cytometry Assay Kit


CountBright™ Absolute Counting Beads

CountBright™ absolute counting beads are a calibrated suspension of microspheres that are brightly fluorescent across a wide range of excitation and emission wavelengths (UV to 635 nm excitation and 385nm emission). CountBright™ absolute counting beads are mixed with the cell sample and assayed via flow cytometry. By comparing the ratio of bead events to cell events, absolute numbers of cells in the sample can be calculated. Because CountBright™ beads are mixed in the test sample, absolute cell counts using this single-platform method are more accurate and less complicated than cell concentration determined using multiple-platform testing. CountBright™ absolute counting beads can be used with any sample type, including no-wash/lysed whole blood.
CountBright 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.

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CountBright™ Absolute Counting Beads