Flow Cytometry Fluorescent Conjugates (Fluorochrome) Optimization

Multicolor panel design tips

  • Always try to pair the brightest fluorochromes with the weakest expressing antigen. In case the expression level of an antigen is unknown, it is advisable to use brighter fluorochromes.
  • Avoid spillover or spectral overlap between fluorochromes by spreading them as much as possible across the spectrum.
  • Avoid fluorochromes that can be excited by more than one laser e.g. APC-Cy7.
  • Always include suitable compensation controls especially if points 3 and 4 above cannot be achieved.
  • It is also advisable to use FMO controls to gate differently stained sub-populations more accurately.
  • Consider using online multicolor panel designers provided by FACS technology companies.

A suitable 4-color panel to illustrate points 1-3. The color of the boxes correspond to the lasers that excite the respective fluorochromes.

Flow Cytometry Multicolor antibody panel

Keywords: FACS fluorophores, fluorochrome conjugates, multi color panel, flow validated antibody panel, excitation emission, FACS dyes