CellTrace™ Reagents for Cell Proliferation
Permanently label cells with CellTrace™ fluorescent stains without affecting morphology or physiology to trace generations or divisions in-vivo or in-vitro.
CellTrace™ Cell Proliferation kits contain a cell membrane–permeable nonfluorescent ester of an amine-reactive fluorescent molecule, which enters cells by diffusion through the plasma membrane. Upon entry into the cell, the previously nonfluorescent molecule is converted to a fluorescent derivative by cellular esterases. The active succinimydyl ester covalently binds to amine groups in proteins, resulting in long-term dye retention within the cell.
Through subsequent cell divisions, each daughter cell receives approximately half of the fluorescent label from the original cell. Each subsequent generation receives half the fluorescence of the parent, 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. 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.
Detecting More Populations with CellTrace™ Violet Stain
Compared to traditional CFSE stains, the CellTrace™ Violet Cell Proliferation Kit has been shown to detect more populations than CFSE or lipophillic stains. One can combine cell proliferation with other live-cell assays.
8 Cell Divisions Detected with CellTrace™ Violet Stain
The CellTrace™ stains are known to be detected after in vitro labeling for several days or approximately 8 division cycles before being overwhelmed by the natural auto fluorescence 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. Stained human lymphocytes. Human CD8+ T lymphocytes stained with 10 µM CellTrace™ Violet followed by incubation in 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.|
Compatiblity with other 488 nm Excited Dyes and Fluorescent Proteins
Since CellTrace™ Violet is excited with violet laser excitation, it can be combined with blue laser–excited dyes (i.e., FITC, Alexa® Fluor 488) and fluorescent proteins (i.e., GFP), to create simple multiparametric panels with minimal compensation.
Using CellTrace™ Violet allows researchers to analyze cell proliferation in combination with other live cell applications (i.e., immunophenotyping, cell sorting, and cell cycle analysis) to maximize the information that can be collected in a single experiment.
Cytotoxicity of CellTrace™ Violet Stain
CellTrace™ Violet stain at recommended concentrations has been shown to exhibit minimal cytotoxic effects on cells. The viability of stained cells was similar to control (untreated) after 7 days in cell culture, and much better than CFSE-stained cells at the same concentration.
|Figure 3. Compatibility of CellTrace™ Violet. Demonstration of the spectral compatibility of CellTrace™ Violet with GFP in cultured osteosarcoma cells that are asynchronous. (A) Unstained cells without GFP expression. (B) Unstained cells stably expressing GFP. (C) 5 µM CellTrace™ Violet–stained cells without GFP expression. (D) GFP-expressing cells stained with 5 µM CellTrace™ Violet.|
|The CellTrace™ CFSE Cell Proliferation Kit provides a versatile and well-retained cell-tracing reagent that is excited by the 488 nm laser on flow cytometers. Carboxyfluorescein diacetate succinimidyl ester, often called CFSE, passively diffuses into cells.|
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|CellTrace™ Far Red DDAO-SE is a fixable, far-red—fluorescent tracer for very long-term cell labeling. This dye is excited by 633/635 nm lasers on flow cytometers. The succinimidyl ester (SE) reactive group forms a strong covalent attachment to primary amines that occur in proteins and other biomolecules on the inside and outside of cells.|
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