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Invitrogen™ CellTrace™ Calcein Red-Orange, AM - Special Packaging

Catalog No. C34851
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C34851 20 x 50 μg
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Catalog No. C34851 Supplier Invitrogen™ Supplier No. C34851
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Description

CellTrace™ Calcein Red-Orange, AM - Special Packaging

CellTrace calcein red-orange AM is a cell-permeant dye that can be used to determine cell viability in most eukaryotic cells. Unlike calcein AM (C-1430, C-3099, C-3100), CellTrace calcein red-orange AM is intrinsically fluorescent; thus, an additional wash step may be necessary to minimize background fluorescence from dye that is not taken up by cells. However, CellTrace calcein red-orange (excitation/emission maxima 577/590 nm) is well-retained by live cells that possess intact plasma membranes, and consequently it is a useful cell tracer and indicator of cell viability.

Specifications

Cell Permeability Cell-permeant
Content And Storage Store in freezer at -5°C to -30°C and protect from light.
Description Calcein, Red-Orange, AM - Special Packaging
For Use With (Application) Cell Tracing, Cell Tracker
For Use With (Equipment) Fluorescence Microscope
Product Type Dye
Dye Type Other Label(s) or Dye(s)
Emission 590
Excitation Wavelength Range 577 nm
Form Lyophilized
Product Line CellTrace
Quantity 20 x 50 μg
Reagent Type Cell Tracker Compounds, Cell Labeling Reagents
Shipping Condition Room Temperature
Target Enzyme Esterase
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Frequently Asked Questions (FAQs)

I need a general cytoplasmic stain that does not overlap with the GFP in my cells. What do you recommend?

Calcein AM, a green dye, is typically used as a general cytoplasmic stain, but not recommended with GFP-positive cells. For GFP-expressing cells there are other colors available: Calcein Blue AM, Calcein Violet AM, and Calcein Red-Orange AM. The retention time of these dyes in live cells is dependent upon the inherent properties of the cell.

How do I reconstitute CellTrace Calcein Red-Orange, AM (Cat. No. C34851)?

You can dissolve the dye using high-quality, anhydrous dimethylsulfoxide (DMSO) up to 1 -2 mg/mL. The acetoxymethyl ester (AM) moiety on CellTrace Calcein Red-Orange, AM is susceptible to hydrolysis when exposed to water absorbed by the DMSO. Once prepared, use the DMSO stock solutions of CellTrace Calcein Red-Orange, AM within a short time period. Aqueous working solutions containing the dye should be prepared fresh and used on the same day. The working principle of CellTrace Calcein Red-Orange, AM is very similar to Calcein, AM, cell-permeant dye (Cat. No. C1430). The protocol given in the product manual for Calcein, AM with an additional wash step is suitable. Wash cells with pre-warmed buffer (e.g. PBS, HBSS) to remove residual serum present in the culture medium, load cells with reagent in buffer, incubate from 15-45 minutes and then wash cells with medium (with or without serum). For further information please see the User Guide.
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 stained my cells with Calcein, AM, but the signal went away after I fixed my cells. Why is this?

Calcein, AM diffuses into cells, the 'AM' moiety is cleaved by cellular esterases, and then the dye molecules are observed in the cytoplasm without binding to anything. This gives a 'whole cell' stain. It also means that the dyes are not crosslinked with aldehyde-based fixation 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 dye from the cell.
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 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.

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