CellTrace™ Calcein Red-Orange, AM - Special Packaging

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.

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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.

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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.

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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.

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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.