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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.

Qtracker™ cell labeling reagents are taken up by the cell through endocytosis and sequestered in endosomes. This gives the label a punctate or vesicular appearance. This is normal. There is nothing that can be done to make it appear uniform throughout the cytoplasm.

Since these dyes insert into lipid membranes, any disruption of the membranes leads to loss of the dye. This includes permeabilization with detergents like Triton™ X-100 or organic solvents like methanol. Permeabilization is necessary for intracellular antibody labeling, leading to loss of the dye. Instead, a reactive dye such as CFDA SE should be used to allow for covalent attachment to cellular components, thus providing for better retention upon fixation and permeabilization.

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 means that they give 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.

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.