Invitrogen™ CellTracker™ Fluorescent Probes

Product Code.	Quantity	Dye Type	unitSize
10724274	5 x 0.1 mg	CellTracker Violet BMQC	500µg
11564237	5 mg	CellTracker™ Blue CMAC	5mg
11574237	5 mg	CellTracker™ Blue CMHC	5mg
11584217	5 mg	CellTracker Blue CMF2HC	5mg
15352644	20 x 15 μg	CellTracker™ Deep Red	300µg
11570166	20 x 50 μg	CellTracker Green CMFDA	1000µg
10644013	1 mg	CellTracker Green CMFDA	1mg
11554237	5 mg	CellTracker Green BODIPY	5mg
11504267	20 x 50 μg	CellTracker Orange CMRA	1000µg
10082742	1 mg	CellTracker Orange CMTMR	1mg
11514267	20 x 50 μg	CellTracker™ Red CMTPX	1000µg

Product Code. 10724274 Supplier Invitrogen™ Supplier No. C10094

Description

CellTracker™ is a fluorescent dye well suited for monitoring cell movement or location. After loading into cells, the dye is well retained, allowing for multigenerational tracking of cellular movements.

Cell movement and location studies require specialized probes that are nontoxic to living cells and well retained, allowing for multigenerational tracking. The CellTracker fluorescent probes are available in a range of fluorescent colors to match instrument lasers and filters, and to accommodate co-staining with antibodies or other cell analysis probes. These dyes are excellent tools for monitoring cell movement, location, proliferation, migration, chemotaxis, and invasion.

Features of the CellTracker dyes include:

• Easy to use—remove culture media, add dye, incubate 15-45 minutes, and image cells
• Excellent retention—fluorescent signal retention of >72 hours (typically three to six generations)
• Ideal tracking dyes—monitor cell movement, location, proliferation, migration, chemotaxis, and invasion
• Low cytotoxicity—does not affect viability or proliferation
• Cell Versatility– works with cells in suspension and adherent cells

The CellTracker fluorescent probes have been designed to freely pass through cell membranes; however, once inside the cell are transformed into cell-impermeant reaction products. The CellTracker fluorescence probes (except for CellTracker Deep Red) contain a chloromethyl or bromomethyl group that reacts with thiol groups, utilizing a glutathione S-transferase–mediated reaction. In most cells, glutathione levels are high (up to 10 mM) and glutathione transferase is ubiquitous. CellTracker Deep Red probe contains a succinimidyl ester reactive group, which reacts with amine groups present on proteins.

After conversion to impermeant versions, the CellTracker fluorescent probes are well retained in living cells through several generations. The probes are transferred to daughter cells, but are not transferred to adjacent cells in a population. Cells loaded with the CellTracker fluorescent probes display fluorescence for at least 72 hours and exhibit ideal tracking dye properties—they are stable, nontoxic at working concentrations, well retained in cells, and brightly fluorescent at physiological pH. Additionally, several CellTracker fluorescent probes with various excitation and emission spectra are available allowing for multiplexing in fluorescence imaging. The CellTracker dyes are retained with fixation and permeabilization, enabling their use with antibodies for fluorescence immunostaining applications.

Spectral characteristics of the fluorescent CellTracker probes:

• CellTracker Blue CMAC—353/466 nm
• CellTracker Blue CMF2HC—371/464 nm
• CellTracker Blue CMHC—372/470 nm
• CellTracker Violet BMQC—415/516 nm
• CellTracker Green CMFDA—492/517 nm
• CellTracker Green BODIPY™—522/529 nm
• CellTracker Orange CMTMR—541/565 nm
• CellTracker Orange CMR—548/576 nm
• CellTracker Red CMTPX—577/602 nm
• CellTracker Deep Red—630/660 nm

Fluorescent CellTracker reagents include the blue-fluorescent chloromethyl derivatives of amino-, hydroxy-, and difluorohydroxycoumarin (CMAC, CMHC and CMF2HC), the green-fluorescent chloromethyl derivatives of fluorescein diacetate (CMFDA) and a BODIPY™ dye, the orange-fluorescent CMTMR and CMRA, and the red-fluorescent CMTPX. CellTracker Blue CMAC, CMHC, and CMF2HC, CellTracker Violet, the violet-fluorescent bromomethyl derivative of coumarin (BMQC), CellTracker Green BODIPY™, CellTracker Orange CMTMR, and CellTracker Red CMTPX do not require enzymatic cleavage to activate their fluorescence, whereas the green CMFDA and orange CMRA do require enzymatic cleavage. The impermeable reaction products of the chloromethyl or bromomethyl coumarins have excellent retention, strong fluorescence, and relatively uniform cytoplasmic staining, making these derivatives potentially useful for correcting motion artifacts in imaging. CMFDA is colorless and non-fluorescent until cytosolic esterases cleave off the acetates, releasing a brightly fluorescent product.

In addition to bright fluorescence and excellent retention, the CellTracker fluorescent probes do not contribute to cytotoxicity.

Order Info

Shipping Condition: Room Temperature

Specifications

• Color: Violet
• Content And Storage: CellTracker™ Violet BMQC, 5 x 0.1 mg vials, requires DMSO
  
  Store at ≤-20°C, protect material from long-term exposure to light; may be exposed to light for short periods of time.
• Excitation Wavelength Range: 415 nm
• Dye Type: CellTracker Violet BMQC
• For Use With (Equipment): Fluorescence Microscope, High Content Instrument
• Quantity: 5 x 0.1 mg
• Detection Method: Fluorescence
• Description: CellTracker™ Violet BMQC Dye, 5 x 0.1 mg
• Emission: 516 nm
• Form: Dry Powder
• Product Line: CellTracker, Molecular Probes
• Reagent Type: Cell Tracker Compounds, Cell Labeling Reagents
• Shipping Condition: Room Temperature
• Label Type: Other Labels or Dyes
• Product Type: Dye
• SubCellular Localization: Cytoplasm

Frequently Asked Questions (FAQs)

I want a dye that can track cells similar to CFSE, but excited with 405 nm violet laser. What is available?

CellTrace Violet and CellTracker Violet BMQC work in the same way as CFSE, but are efficiently excited with the violet laser. They are comparable to CFSE in terms of cell retention, and has been used for tracking cells with microscopy as well as flow cytometry.

I want to do a cell migration study for around 4 hours and need to fluorescently label the cells with a dye. What do you recommend?

Calcein, AM and FDA (fluorescein diaceate) are examples of some dyes used for this application. Since these dyes are not incorporated or covalently attached to any cellular components, they may have a short retention time as some cell types may actively efflux the dye out of the cells. The CellTracker and CellTrace dyes include either a mild thiol-reactive chloromethyl group or amine-reactive succinnimidyl ester group to allow for covalent binding to cellular components, providing for better retention. As with any reagent, one should empirically determine retention times for the cell type used.

I labeled my cells with Calcein, AM, but when I imaged the next day, there was no fluorescence from Calcein. Why?

Calcein, AM is a good choice for cell tracking and as a general cytoplasmic stain. However, it doesn't bind to anything and may be actively pumped out of the cells within a couple hours, which is likely what happened. The retention of Calcein within live cells is dependent upon the inherent properties of the cell type and culture conditions.

For long-term imaging, you may wish to consider a reactive cytoplasmic stains such as CFDA, SE or the CellTracker and CellTrace dyes.

Can the CellTracker dyes be fixed?

Yes, the CellTracker dyes react with any accessible thiol part of the protein and can be fixed. However, some CellTracker dyes may be attached to small metabolites that can leak from the cell following permeabilization. This can result in decreased fluorescence.

I stained two populations of cells, one with CellTracker Green and the other with CellTracker Red, but it looks like there may be crossover of the red dye to the green cells. What is going on?

One possibility is that there is spectral bleedthrough between the dyes. Be sure to check the single-color samples by imaging the red cells in green and imaging the green cells in red, using the optimal imaging settings for the other color. If you see bleedthrough with these controls, then you will have to reduce the dye label concentration to reduce the brightness of the dyes, or choose dyes that are farther apart spectrally. If the issue isn’t bleedthrough, another possibility is that the cells were not adequately washed after staining, allowing some unincorporated dye to remain and label the other cells after they were introduced. Extending washes and wash times should help with this.

I'm trying to stain my cells with CellTracker dyes or CFDA SE, but I'm not seeing much signal. What can I do?

First, make sure you aren’t staining in the presence of serum, since serum can have esterase activity that can prematurely cleave the AM group on these dyes, preventing entry into cells. After staining, it’s okay to return the cells to medium containing serum. After this, you can try increasing the concentration and label time to get a higher intensity.

I like how calcein dyes label the whole cell. How long can I track my cells with them, and can I fix them?

Calcein dyes diffuse into cells, the 'AM' moiety is cleaved by cellular esterases and then are observed in the cytoplasm without binding to anything. This provides a 'whole cell' label. Calcein dyes may be pumped out by normal cellular efflux mechanisms, sometimes within a very short time, especially for cell types that may exhibit drug resistance, unless the efflux is inhibited (such as with probenecid). The dyes are not crosslinked with aldehyde-based fixation, unlike protein-binding CellTracker dyes, and thus will be lost upon fixation. Additionally, any disruption of plasma membrane, such as with detergents or trypsinization, will lead to leakage of the dyes from the cell.

I want to track my cells with a nucleic acid stain, like DAPI or Hoechst dye. Do you recommend this?

This is not recommended. When these stains bind to DNA and RNA, they may affect the normal function of the nucleic acids, disrupting transcription, as well as replication. Other reagents, such as CellTracker dyes or Qtracker reagents are more optimized for tracking without disrupting normal activity. If a nuclear label is still desired, though, and the cells are mammalian and non-hematopoietic, CellLight nuclear reagents can transiently transfect cells to express GFP or RFP on a nuclear-expressing protein for up to several days without affecting function.

I want to track my cells over time, and you have a lot of options to choose from. How do I pick the right one?

Please see this Web link (http://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-tracing-tracking-and-morphology/cell-tracking.html) to help you choose the right option for your application. Start by planning how long you want to track your cells, then consider the mechanism of binding. Calcein dyes are very uniform in label and are good for short-term cell migration, but may be rapidly effluxed from some cell types. Lipophilic cyanine dyes, such as DiI, DiO, and similar dyes label cell membranes, don’t disrupt function, and can last longer, but have the potential to cross to other cells if membranes fuse. They are also lost upon permeabilization. CellTracker dyes are better for longer-term labeling, as they possess a mildly reactive chloromethyl moiety that allows covalent binding to cellular components. CFDA SE also covalently binds to cellular components. With all the reagents, their retention within cells is dependent upon the rate of cell division and the inherent properties of the cell (active efflux, membrane and protein turnover rates, etc.) and reagents that allow for covalent attachment exhibit longer retention than those that do not.

The longest-lasting and brightest options are the Qtracker reagents, which are taken up through endocytosis. These are so bright individual quantum dots can be detected, and are also robust enough to survive not only fixation and permeabilization, but even the heat and solvents used in paraffin processing.

For Research Use Only. Not for use in diagnostic procedures.