CellTrace CFSE Cell Proliferation Kit Protocol

The CellTrace CFSE Cell Proliferation Kit provides a robust method for evaluating cell proliferation through CFSE staining. CFSE, or carboxyfluorescein succinimidyl ester, is a fluorescent dye used in CFSE proliferation assays to monitor distinct generations of proliferating cells. The CFSE staining protocol involves covalently labeling live cells with this bright and stable dye, which dilutes evenly among daughter cells during cell division. Flow cytometric visualization then allows researchers to observe each generation as a separate peak on a flow cytometry histogram, offering precise insights into cell proliferation dynamics. The CellTrace CFSE protocol is essential for studies requiring accurate measurement of cell proliferation and is widely used in various research applications to evaluate cell division and growth.

See all cell proliferation assays for flow cytometry

On this page:

Materials
Protocol
Ordering information
FAQs
Resources

Materials

This protocol can be used for:

Detecting cell proliferation using flow cytometry

This protocol should not be used for:

Fluorescence microscopy or microplate readers

You will need the following for this protocol:

20 mL of heparinized peripheral whole blood
CellTrace CFSE Cell Proliferation Kit
OpTmizer T Cell Expansion SFM
Penicillin-Streptomycin-Glutamine (100X)
PBS
Ficoll-Paque Plus
Cell counter such as the Countess 3 FL
Dynabeads Human T-Activator CD3/CD28

Top

Protocol steps

Culture medium preparation

To 1 L Gibco OpTmizer T Cell Expansion SFM, add the following:
- 26 mL of T Cell Expansion Supplement
- 10 mL of Penicillin-Streptomycin-Glutamine
Complete medium is stable for 4 weeks when stored at 2–8°C in the dark.

Mononuclear cell isolation from whole blood

Dilute 10 mL whole blood in 10 mL PBS and mix well.
Add 15 mL Invitrogen Ficoll-Paque Plus to a 50 mL centrifuge tube and gently layer 20 mL diluted whole blood on top.
Centrifuge for 30 minutes at 400 x g.
Carefully remove lymphocyte layer and transfer to a new tube.
Resuspend cells in 25 mL DPBS buffer in a 50 mL conical tube.
Centrifuge for 5 minutes at 300 x g, pour off supernatant, and resuspend in 25 mL DPBS.
Repeat wash step and resuspend in 10 mL DPBS.
Count cells on the Invitrogen Countess Automated Counter or by another method; adjust concentration to 10⁶ cells/mL.

Cell staining

Add 18 µL DMSO to a vial of CellTrace CFSE staining solution.
Dilute this stock solution into 20 mL of PBS (warmed to 37°C) for a 5 µM staining solution.
Add 10 mL of cells to a 50 mL centrifuge tube.
Centrifuge cells for 5 minutes at 300 x g and carefully pour off supernatant.
Resuspend cells in 10 mL of CellTrace CFSE staining solution.
Incubate cells for 20 minutes in a 37°C water bath.
Add 40 mL OpTmizer T Cell Expansion SFM to the cells to absorb any unbound dye.
Incubate cells for 5 minutes.
Centrifuge cells for 5 minutes at 300 x g and resuspend the cell pellet in pre-warmed OpTmizer T Cell Expansion SFM.

Stimulation and analysis

Distribute aliquots of stained cells into culture plates or flasks.
Stimulate with 50 µL Invitrogen Dynabeads Human T-Activator CD3/CD28 per 1 mL of cells, or other stimulus.
Incubate for desired length of time under growth conditions.
Harvest cells and stain for other markers if appropriate.
Analyze using a flow cytometer with 488 nm excitation and emission filters appropriate for fluorescein.

Spectral information and storage

	CellTrace CFSE
Excitation/Emission (in nm)	492/517
Standard filter set	Invitrogen Alexa Fluor 488
Storage conditions	≤–20°C

Protocol tips

Reserve 1 mL of cells for unstained control and 1 mL of cells for a stained, but unstimulated control
Dynabeads stimulation typically results in T cell division every 18–20 hr
Analyze as many cells as possible from each sample
Use a viability dye and gate on live cells

Top

Ordering information

CellTrace CFSE materials

Frequently asked questions

How does CellTrace dye work?

CellTrace dyes, such as CellTrace CFSE dye, label cells by covalently binding to intracellular proteins. Here’s a brief overview of the process:

Preparation: The dye is reconstituted in a solvent like DMSO.
Labeling: The dye solution is added to live cells, where it diffuses into the cytoplasm.
Binding: The dye reacts with amine groups on proteins, forming stable covalent bonds.
Fluorescence: Labeled cells fluoresce brightly when excited by specific wavelengths (495 nm for CellTrace CFSE dye).
Tracking: As cells divide, the dye is equally distributed, halving the fluorescence intensity with each division. This allows tracking of cell proliferation using flow cytometry or microscopy.
Fixation: Cells can be fixed with aldehyde fixatives (e.g., paraformaldehyde) to preserve fluorescence for analysis.

What is CFSE staining?

Carboxyfluorescein succinimidyl ester (CFSE) staining is a fluorescent cell labeling technique used to track cell division and proliferation. CFSE is a cell-permeable dye that covalently binds to intracellular proteins, resulting in long-lasting fluorescence. As cells divide, the dye is equally distributed between daughter cells, allowing researchers to monitor cell division over time by measuring the dilution of fluorescence intensity.

What is the color of CFSE stain?

The color of CFSE stain is green. When excited by a laser (typically at 488 nm), CFSE emits fluorescence with a peak around 517 nm, which appears green. This makes it suitable for use in flow cytometry and fluorescence microscopy to track and analyze cell proliferation.

How does CFSE compare to BrdU?

CFSE is often preferred for long-term studies and for its ability to provide quantitative data on cell division through fluorescence intensity. BrdU is useful for short-term studies and specifically for identifying cells in the S-phase of the cell cycle.

Feature	CFSE (carboxyfluorescein succinimidyl ester)	BrdU (bromodeoxyuridine)
Labeling mechanism	Covalently binds to intracellular proteins	Incorporates into newly synthesized DNA
Detection method	Fluorescence (green)	Colorimetric or fluorescence (dependent on label used)
Cell division tracking	Tracks cell division by fluorescence dilution	Tracks cell proliferation by DNA incorporation
Duration of labeling	Long-term (up to several weeks)	Short-term (hours to days)
Compatibility	Flow cytometry, fluorescence microscopy	Flow cytometry, immunohistochemistry
Cell viability	Generally non-toxic	Can be toxic at high concentrations
Quantitative analysis	Yes, through fluorescence intensity	Yes, through antibody staining
Applications	Cell proliferation, migration studies	Cell proliferation, S-phase detection

Top

Resources

Cell Viability, Proliferation, and Cell Cycle Information
Find educational resources for monitoring cell function

Cell Proliferation Assay Protocols
Discover protocols for various applications to study cell proliferation.

Cell Cycle & Proliferation Pathways
Investigate signaling pathways and signal transduction.

Fluorescence SpectraViewer
Use SpectraViewer to easily compare excitation and emission spectra of fluorophores and reagents.