Calcein AM, Cell-permeant Green Dye, 1 mL - FAQs

View additional product information for Calcein AM, Cell-permeant Green and Blue Dyes - FAQs (C1430, C3100MP, C481, C3099, C1429, C34852)

11 product FAQs found

I would like a dye to load in live cells such that it will self-quench at a high concentration, but if the cell dies, the dye will be released and unquenched. Do you have anything like that?

Yes. This is commonly done with calcein AM or FDA (fluorescein diacetate). These dyes will not fluoresce until cleaved by esterases. After modification by esterases and at very high concentrations, they will self-quench. Upon disruption of the plasma membrane, or cell death, the dye will be released into the extracellular medium, and become unquenched. Concentration and incubation time must be optimized to obtain adequate quenching.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

I need to load liposomes with calcein. Should I use the AM form or the non-AM form?

Calcein, AM requires esterase cleavage of the acetoxymethyl (AM) ester to become fluorescent. Liposomes don't have esterases unless specifically constructed to include the enzyme. The water-soluble, non-AM form of calcein (Cat. No. C481), does not require esterase cleavage to be fluorescent.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

I am doing a Live/Dead assay using Calcein, AM, for live cells and ethidium homodimer-1 for dead cells. Can I fix the cells after labeling and retain the staining?

This is not recommended. Neither Calcein nor ethidium homodimer-1 bind to any cellular components upon fixation. There is no guarantee that the dyes will be retained upon fixation or any subsequent wash steps. We recommend scoring for live and dead cells as soon as possible after staining.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

I wish to assay the viability of my cells over several days. Can I use any of your LIVE/DEAD imaging kits to do this?

No, it is not possible to assay the viability of the same population of cells longer than a few hours; you will need to use replicate samples. DNA binding dyes are toxic to cells; stained cells should be imaged as soon as possible after staining. Calcein AM is not retained in cells and may be actively effluxed out in the range from minutes to several hours, dependent upon the cell type. Calcein AM is not toxic to cells, so it can be added repeatedly to the same samples. You can assay the proliferation of the same sample of cells over several days using alamarBlue reagent or PrestoBlue reagent, as these dyes are non-toxic to cells.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Cell Tracing and Tracking Support Center.