What guidelines should I follow for successful tandem dye usage?

Using tandem dyes in flow cytometry can significantly enhance the capabilities of multicolor staining and allow for the detection of multiple markers simultaneously. To ensure successful tandem dye usage, consider the following guidelines:

1. Avoid extended exposure to light, tandem dyes are high susceptible to photobleaching and should be protected from light at all times.
2. Do not freeze tandem conjugates, this can result in the denaturing of the donor
3. Be cautious, fixation and permeabilization steps can reduce tandem dye brightness. If a fixation or permeabilization step is required, keep the duration short and mild.
4. Use appropriate controls, such as isotype controls for background, unstained cells and single color controls for compensation when performing multicolor flow cytometry
5. Determine if compensation is necessary before analyzing data, this is due to two or more tandems with overlapping emission spectra