"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 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 succinimydyl 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.
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).
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.
More flexibility in multiplexed experiments
The CellTrace Cell Proliferation Kits are available for the 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 (Figure 4) 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 3. Generational tracing using CellTrace reagents. Cell proliferation was followed for varying numbers of generations under different experimental conditions using (A) CellTrace Violet reagent in human peripheral blood mononuclear cells (PBMCs) stimulated for 7 days; (B) CellTrace CFSE reagent in human T lymphocytes stimulated for 5 days; (C) CellTrace Yellow reagent in human PBMCs stimulated for 11 days; and (D) CellTrace Far Red reagent in human T lymphocytes stimulated for 5 days. PBMCs were stimulated with anti-human CD3 antibody (Cat. No. MHCD0300) and interleukin-2 (Cat. No. PHC0027) while human T lymphocytes were stimulated with anti-human CD3 antibody (Cat. No. MHCD0300) alone. Unstimulated parent generations are represented by (A) red peak; (B) blue peak and (C) and (D) purple peak. Dead cells were excluded from each data group using SYTOX dead cell stains. Analyses performed using an Attune Flow Cytometer for three of the reagents with the following excitation: bandpass emission filter for each: 405 nm: 450/40 nm for CellTrace Violet detection; 488 nm: 530/30 nm for CellTrace CFSE detection; and 638 nm: 660/20 nm for CellTrace Far Red detection. CellTrace Yellow reagent analysis was performed on the Attune NxT Flow Cytometer using excitation at 561 nm and detection with 585/16 nm bandpass emission filter.
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.