Carboxyfluorescein succinimidyl ester, or CFSE, is a fluorescent dye used by scientists to monitor cell division, a procedure known as a cell proliferation assay. It allows researchers to track how many times a cell has divided over a period of time. This is accomplished by measuring the amount of dye within a cell population. Invitrogen, a brand under Thermo Fisher Scientific, is a supplier of a widely used kit for this purpose, the CellTrace™ CFSE Cell Proliferation Kit, which provides a reliable method for labeling cells to trace multiple generations.
Mechanism of CFSE Cell Labeling
CFSE’s function begins with its form as carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), a non-fluorescent molecule that can pass through a cell’s membrane. Once inside the cell, intracellular enzymes called esterases cleave off acetate groups. This enzymatic reaction transforms the molecule into the highly fluorescent CFSE. This process also traps the dye, preventing it from leaking back out of the cell.
With the CFSE molecule activated and contained within the cell’s cytoplasm, its succinimidyl ester component comes into play. This part of the dye forms strong, covalent bonds with primary amines, which are found on proteins throughout the cell’s interior. This creates a stable fluorescent label attached to the cell’s internal protein machinery. Because these bonds are covalent, the dye is well-retained and does not transfer to neighboring cells.
The principle that allows CFSE to track cell division is based on the distribution of these labeled proteins. When a cell divides into two daughter cells, its components, including the CFSE-labeled proteins, are shared roughly equally between them. Consequently, each daughter cell inherits about half the fluorescence of the parent cell. This predictable dilution of the fluorescent signal allows researchers to count how many times cells in a population have divided.
Protocol for Staining Cells with Invitrogen CFSE
The first step of the Invitrogen CellTrace™ CFSE protocol involves preparing the cells. It is necessary to begin with a healthy, actively dividing cell population that has been prepared as a single-cell suspension. The concentration of cells should be optimized, as this can influence staining efficiency and subsequent analysis.
Next, the CFSE dye is prepared for use. The Invitrogen kit provides the dye in a dry, single-use format to ensure consistency. This dry dye is first dissolved in a small amount of anhydrous dimethyl sulfoxide (DMSO) to create a concentrated stock solution. This stock solution is then diluted into a pre-warmed buffer, such as phosphate-buffered saline (PBS), to create the final working solution.
With the cells and dye ready, the staining process begins. The prepared cell suspension is mixed with the CFSE working solution and incubated to allow the dye to enter the cells and bind to intracellular proteins. Gentle agitation during this incubation can help ensure uniform labeling across the entire cell population.
Following incubation, the staining reaction must be stopped and any unbound dye removed. This is achieved by adding a large volume of complete culture medium, which quenches the reactivity of free CFSE. The cells are then centrifuged to form a pellet, the supernatant is discarded, and the cell pellet is resuspended in fresh culture medium. Thorough washing is necessary for reducing background fluorescence that could interfere with data interpretation.
Analyzing CFSE Data with Flow Cytometry
After the cells have been stained and cultured, the results are analyzed using flow cytometry. This instrument measures the fluorescence of individual cells as they pass one by one through a laser beam. The output is displayed as a histogram, where the x-axis represents fluorescence intensity and the y-axis represents the number of cells.
To properly interpret this data, control samples are necessary. An unstained cell sample is used to measure baseline autofluorescence, the natural fluorescence of the cells. This allows the researcher to set a gate that distinguishes stained cells from the background. A second control is a sample of cells stained with CFSE but not stimulated to divide, called the “Day 0” or parent generation, which defines the fluorescence intensity of the undivided population.
As the stained cells divide, the fluorescence is halved with each generation. This appears on the flow cytometry histogram as a series of distinct peaks to the left of the parent peak. Each peak represents a successive generation of cells, with an intensity approximately half that of the peak to its right. A bright initial stain makes it possible to resolve up to eight or more distinct generations before the signal becomes indistinguishable from autofluorescence.
Specialized software is then used to analyze the distribution of cells across these generational peaks. From the histogram, metrics can be calculated to quantify proliferation. Common parameters include the proliferation index, which is the average number of divisions for all cells in the original population, and the division index, the average number of divisions for only the cells that divided.
Common Applications and Optimization
CFSE’s ability to track cell division has made it a tool in various fields of biological research. In immunology, it is used to monitor the activation and proliferation of lymphocytes, such as T cells, in response to stimuli. Cancer research utilizes CFSE assays to measure the proliferative rates of tumor cells and to assess the effectiveness of anti-cancer drugs. The dye’s stability also allows for in vivo studies, where labeled cells are injected into an organism to track their migration and division.
For a CFSE assay to yield reliable data, optimization is required. A common issue is cytotoxicity; a CFSE concentration that is too high can be toxic to cells, inhibiting the proliferation the experiment is designed to measure. Finding a balance where the dye is bright enough to resolve multiple generations without harming the cells is a primary goal.
Another potential problem is poor peak resolution in the flow cytometry data, where generational peaks overlap. This can be caused by inconsistent staining, variability in cell division times, or inadequate washing after the staining step. Performing a titration experiment to identify the optimal dye concentration for a specific cell type is an effective way to address many of these issues.