Invitrogen™ CellTrace™ Violet Cell Proliferation Kit, for flow cytometry

Catalog No.	Quantity
50-112-1516	20 Assays
C34557	180 Assays

Catalog No. 50-112-1516 Supplier Invitrogen™ Supplier No. C34571

Description

CellTrace™ Violet Cell Proliferation Kit, for flow cytometry

CellTrace™ Violet Cell Proliferation Kit is used for in vitro and in vivo labeling of cells to trace multiple generations using dye dilution by flow cytometry.

Superior performance—bright, single-peak staining enables visualization of multiple generations

Long-term signal stability—well-retained in cells for several days post stain

Versatile—multiple colors available to easily combine with antibodies or markers of cell function, such as GFP

Simple, robust staining protocol

View a selection guide for all CellTrace™ Cell Proliferation Kits for flow cytometry.

Superior fluorescent staining
Successful proliferation analysis by dye dilution (see figure below) requires an extremely bright dye to distinguish fluorescently labeled cells from auto-fluorescence after several cell divisions. The intense fluorescent staining provided by CellTrace™ Violet dye enables the visualization of ten or more generations of proliferating cells before the signal is overwhelmed by intrinsic cellular auto-fluorescence. Consistent, homogeneous staining results in very little fluorescence variation between cells in a population, so distinct generations can be seen without any requirement for complex modeling software.

Long-term signal retention
Unlike stains that label the lipid membrane of cells, CellTrace™ Violet stain easily crosses the plasma membrane and covalently binds inside cells where the stable, well-retained fluorescent dye provides a consistent signal, even after several days in a cell culture environment. CellTrace™ Violet dye binds covalently to all free amines on the surface and inside of cells and shows little cytotoxicity, with minimal observed effect on the proliferative ability or biology of cells.

Easy multiplexing with other fluorophores
The violet excitation and narrow emission of CellTrace™ Violet dye make it ideal for multiplexing due to the limited spectral overlap with other common dyes (Alexa Fluor™ 488, FITC, and RPE) and fluorescent proteins (Green Fluorescent Protein (GFP) and mCherry) (see Fluorescence Spectra for CellTrace™ Violet stain below).

Simple, robust staining protocol
The CellTrace™ Violet Cell Proliferation Kit contains convenient single-use vials of dry dye to permit small-scale experiments without preparing excess quantities of dye. A stock solution is prepared by dissolving the contents of a vial in anhydrous DMSO prior to use. To stain 1 mL of cells in protein-free medium, 1μL of this stock solution is typically used. Cells should be stained for 20 minutes at room temperature with gentle agitation. A brief wash with complete medium will then quench any dye remaining in solution.

Specifications

• Content And Storage: Contains 1 vial of CellTrace™ Violet reagent and 1 vial of DMSO (100 μL).
  Store in freezer -5°C to -30°C and protect from light.
• Detection Method: Fluorescence
• For Use With (Application): Proliferation Assay
• For Use With (Equipment): Flow Cytometer
• Product Type: Cell Proliferation Kit
• Dye Type: Contains 1 vial of CellTrace™ Violet reagent and 1 vial of DMSO (100μL)
• Emission: 405/450
• Form: Solid
• Format: Tube(s)
• Product Line: CellTrace
• Quantity: 20 Assays
• Reagent Type: CFSE & Related Compounds
• Shipping Condition: Room Temperature
• Solubility: DMSO (Dimethylsulfoxide)

Frequently Asked Questions (FAQs)

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.

How should I prepare the stock solution for CellTrace Violet Cell Proliferation Kit, for flow cytometry (Cat. No. C34557, C34571)?

To prepare a stock solution of CellTrace Violet Cell Proliferation Kit, for flow cytometry (Cat. No. C34557, C34571), dissolve the contents of one vial in 20 µL of anhydrous DMSO, just prior to use, to give a stock concentration of 5 mM. The recommended working concentration is 5 µM.

I would like to label two cell populations with two different CellTrace reagents and then co-culture these cells. Will the CellTrace reagent leave the cells to stain other cells?

We have not tested the use of the CellTrace reagents for co-culture applications. In theory, this may work, but you would have to test this on your cells of interest.

With the CellTrace cell proliferation kits, for flow cytomtery, I am getting only one broad peak on my histogram instead of multiple peaks. What is causing this?

A single broad peak is usually caused by using too high a concentration of dye and/or too long an incubation time.

What cellular components do the CellTrace Proliferation dyes label?

The dyes provided in the CellTrace Proliferation kits are amine-reactive dyes that are considered general cytoplasmic stains. They may bind to various membrane proteins, organelles as well as components in the cytoplasm. They are not known to localize in any specific organelles.

I am storing the DMSO stock solutions of the CellTrace cell proliferation reagents and am not obtaining a good signal with my cells compared to freshly dissolved dye stock. Why does the product not work after storage?

We provide the CellTrace reagents in small aliquots and strongly recommend discarding any unused DMSO/dye stocks. The CellTrace reagents have acetyl groups to cap the charges on the dyes to make them cell permeant, and succinimidyl ester amine-reactive moiety to allow for covalent attachment to cellular components for long-term retention. Both acetyl groups and succinimidyl esters will readily hydrolyze if any water is present during storage. DMSO is hygroscopic and thus readily absorbs water from the atmosphere. If you must store your dye stocks, you will need to use a good quality, anhydrous DMSO stock that has not been opened often and store the vial within an air-tight container containing some desiccant to keep the DMSO/dye stock solution anhydrous during storage.

I am using the CellTrace Cell Proliferation reagents and am not obtaining good separation of my cell generation peaks. How can I improve the peak separation?

Here are some tips to obtain uniform staining and a bright, unstimulated parent generation peak:

Dissolve the CellTrace dye stock immediately before use in the DMSO provided in the kit or in good quality, anhydrous DMSO to obtain the best reactivity and cell permeability.
Stain in PBS or other amine-free, protein-free physiological buffer. Do not stain in medium.
Start with a single-cell suspension and gently agitate the cells during staining.
Quickly remove the unbound dye by incubating the cells in ice-cold media for 5 minutes and then wash twice more with pre-warmed media.
Include a dead-cell stain in the assay and gate only on live cells.
Analyze as many cells as possible from each sample.
Use a low flow rate for analysis on hydrodynamic focusing cytometers.
A good staining concentration for the CellTrace dyes is generally within 1-10 µM, but the optimal concentration for a particular cell type will vary. Observe your cells in a stain dilution series to determine the optimal concentration for your cells.
Some cell types may take up dye with a broad staining intensity distribution. If this is the case for your cells, then you will need to do an initial sort of the stained, unstimulated parent cells to select for a narrow peak distribution.

Can I store the stock solutions of the CellTrace reagents and how do you recommend storing them?

We provide the CellTrace reagents in small aliquots and strongly recommend discarding any unused DMSO dye stocks. Some of the CellTrace reagents have diacetate groups to cap the charges on the dyes to make them cell permeant, and some have succinimidyl ester amine-reactive groups for long-term cellular retention. Both diacetates and succinimidyl esters will readily hydrolyze if any water is present during storage. DMSO is very hygroscopic and thus readily absorbs water from the atmosphere. We do not recommend storing stock solutions of CellTrace reagents because storage of the product in solution will inevitably lead to partial or complete loss of reactivity.

How do the CellTrace Cell Proliferation reagents work?

CellTrace Cell Proliferation reagents are all cell-permeant dyes that are cleaved by intracellular esterases to yield highly fluorescent compounds that also covalently bind to cellular amines, attaching the dye to various cellular components and providing a very stable signal. These reagents show little cytotoxicity with minimal observed effects on the proliferative ability of many cells.

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.

I am trying to assay cell proliferation with a CellTrace stain, and I am not seeing separate peaks for each cell division. How can I optimize this assay?

The key to good generational profiles with CellTrace reagents is starting with cells that are evenly labeled so that they have a tight coefficient of variance (CV) when run at time zero after labeling. If the peak is too broad, the generations will overlap each other and the series of peaks will become a hump. Even labeling can be achieved by starting with a uniform cell population (not a mixture of lymphocytes and granulocytes for example) as staining will be proportional to cell size. Cells are labeled rapidly, so you want to pre-dilute the dye and mix it into your cells rapidly. Be sure that the cells are not sitting in a clump in the bottom of your tube. The easiest way to do this is to make a 2x dye solution (1x = 1-10 µM) and resuspend your cells in a half volume of medium (no serum or BSA). Add the dye to the cells and invert a few times to mix. Gently agitate the cells during staining. Once the dye incubation is over (20 min, 37 degrees C), add serum or BSA (at least 1%) to scavenge any remaining unreacted dye. Spin down cells, wash 1x, and resuspend in complete medium. After a 10-20 min incubation to undergo de-esterification, cells are ready to be set up for whatever treatment you are planning. Be sure to keep a time zero control as you need to know where the first generation ran.

What are the advantages of flow cytometry?

-Measures data from single cells.
-Data are obtained for a large number of cells, generating a rich statistical analysis of cell populations.
-Because single cells are measured, it will reveal heterogeneity within a population.
-With the ability to multiplex, small sub-populations can be identified.
-Thousands of cells can be analyzed rapidly.
-It is ideally suited for blood samples and other cells in suspension.
-Data can be re-analyzed multiple times after acquisition.
-Flow cytometry files (FCS) can be archived.

What kinds of applications can I run on a flow cytometer?

There are several applications, some of which include immunophenotyping, cell cycle analysis, apoptosis assays such as annexin V staining, CellEvent Caspase-3/7 assay, and TUNEL assay, cell viability, proliferation assays such as CellTrace assay and Click-iT EdU assay, measurements of mitochondrial potential with MitoProbe assays, and cell counting using counting beads.

Safety and Handling

WARNING: Reproductive Harm - www.P65Warnings.ca.gov

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