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 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.
