Cartoon of dye dilution experiment showing first generation as the brightest. Each subsequent generation is half as bright.

Permanently label cells with Invitrogen CellTrace fluorescent stains without affecting morphology or physiology to trace generations or divisions in vivo or in vitro.

  • Superior performance—bright, single-peak staining enables visualization of multiple generations
  • Long-term signal stability—well retained in cells for several days post-stain
  • Non-cytotoxic—no known effect on proliferative ability or biology of cells
  • Versatile—multiple colors available to easily combine with antibodies or markers of cell function, such as GFP

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"CellTrace Violet is the best reagent for tracking proliferation in any amenable cell type by fluorescent dye dilution and flow cytometry. Compared to CFSE which is cytotoxic to cells when used at higher concentrations, CellTrace Violet labels cells brightly, with low toxicity and is faithfully distributed to daughter cells ensuring the best possible peak resolution.”

—Andrew Filby, Flow Cytometry Core Facility Manager and ISAC SRL Emerging Leader,
Newcastle University

CellTrace Cell Proliferation Kit selection guide

Far Red 
Basis of assayPermanently label cells with fluorescent stains to trace generations or divisions in vivo or in vitro without affecting morphology or physiology.
ReadoutThe level of fluorescence in the cell populations determines the number of generations through which a cell has progressed since the fluorescent label was applied.
Fluorescent label
Far Red
LaserUV405488532, 561633/635
Ex/Em (nm)355 or 375/410405/450495/519546/579630/661
20 tests
180 tests

How CellTrace Cell Proliferation Kits work

CellTrace Cell Proliferation Kits contain a cell-permeant nonfluorescent ester of an amine-reactive fluorescent molecule, which enters cells by diffusion through the plasma membrane. Upon entry into the cell, the nonfluorescent molecule is converted to a fluorescent derivative by cellular esterases. The active succinimidyl ester covalently binds to amine groups in proteins, resulting in long-term dye retention within the cell.

Through subsequent cell divisions, daughter cells receive approximately half of the fluorescent label of their parent cells, allowing the analysis of the fluorescence intensities of cells labeled and grown in vivo.

Analysis of the level of fluorescence in the cell populations by flow cytometry permits the determination of the number of generations through which a cell has progressed since the label was applied (Figure 1).

How CellTrace Cell Proliferation Kits work

Figure 1. Mechanism of cell labeling. (A)
Illustration of proliferation analysis by dye dilution. (B) Flow cytometric analysis reveals a bright, homogenous fluorescent signal from the initial population of cells. Subsequent cell divisions result in larger numbers of cells, each with half the fluorescence intensity of its parent cell.

To understand more about the suitability of these reagents for tracking proliferation as compared to lipophilic fluorescent dyes, Filby and coworkers published a method to assess and compare the performance of three succinimidyl ester–based dyes (including Invitrogen CellTrace Violet and CellTrace Far Red dyes) and two lipophilic dyes.

Article: Appraising the Suitability of Succinimidyl and Lipophilic Fluorescent Dyes to Track Proliferation in Non-Quiescent Cells by Dye Dilution

Sensitive detection of cell populations

The CellTrace stains have been detected after in vitro labeling for several days, or approximately 7–8 division cycles, before being overwhelmed by the natural autofluorescence of the cells. Up to 10 cell divisions have been observed using the CellTrace Violet Cell Proliferation Kit on a violet laser–equipped flow cytometer (Figure 2).

Article: A comparison study of CellTrace Violet, Yellow, and Far Red

Sensitive detection of cell populations

Figure 2. Stained human lymphocytes. Human CD8+ T lymphocytes stained with 10 µM CellTrace Violet followed by incubation in Gibco OpTmizer T cell Expansion Medium at 37°C for 7 days. Cells were stimulated with 200 ng mouse anti-human CD3 antibody and 100 ng interleukin-2 per milliliter cells. Analysis of the CellTrace data was done using ModFit LT™ software from Verity Software House, which uses modeling to define generational populations, as shown in the figure.

More flexibility in multiplexed experiments

The CellTrace Cell Proliferation Kits are available for the UV, violet, blue, yellow (561 nm), and red lasers (Figure 3), allowing researchers to analyze cell proliferation in combination with other live-cell applications (i.e., immunophenotyping, cell sorting, and cell-cycle analysis), maximizing the information that can be collected in a single experiment, with minimal compensation.

CellTrace Violet and CellTrace Far Red are also compatible with commonly used green-fluorescent dyes like FITC and Invitrogen Alexa Fluor 488, or with green fluorescent proteins (i.e., GFP) (Figure 4).

Poster: Multiplexing CellTrace Violet with common flow cytometry reagents
Poster: Multiplexing CellTrace Yellow with common flow cytometry reagents
Article: Multiplexing CellTrace Far Red with GFP

Generational tracing using CellTrace reagents
Figure 3. Generational tracing using CellTrace reagents. Cell proliferation was followed for varying numbers of generations under different experimental conditions using (A) CellTrace Blue reagent, (B) CellTrace Violet reagent, (C) CellTrace CFSE reagent, (D) CellTrace Yellow or (E) CellTrace Far Red reagent.
Compatibility of CellTrace Violet stain.

Figure 4. Compatibility of CellTrace Violet stain. Demonstration of the spectral compatibility of CellTrace Violet with GFP in cultured asynchronous osteosarcoma cells. (A) Unstained cells without GFP expression. (B) Unstained cells stably expressing GFP. (C) Cells stained with 5 µM CellTrace Violet stain, without GFP expression. (D) GFP-expressing cells stained with 5 µM CellTrace Violet stain.

Featured video

Multiplexing with the CellTrace Violet dye cell proliferation reagent

Dr. Andrew Filby, Ph.D. Flow Cytometry Core Facility Director at Newcastle University in the UK, shares his experience multiplexing the CellTrace Violet reagent as compared to CFSE to trace multiple generations by dye dilution using flow cytometry.


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