Permanently label cells with Invitrogen CellTrace fluorescent stains without affecting morphology or physiology to trace generations or divisions in vivo or in vitro.
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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.”
|Basis of assay||Permanently label cells with fluorescent stains to trace generations or divisions in vivo or in vitro without affecting morphology or physiology.|
|Readout||The level of fluorescence in the cell populations determines the number of generations through which a cell has progressed since the fluorescent label was applied.|
|Ex/Em (nm)||355 or 375/410||405/450||495/519||546/579||630/661|
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).
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.
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).
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.
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).
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.
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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For Research Use Only. Not for use in diagnostic procedures.