Polychromatic Flow Cytometry

The ability to measure multiple parameters of individual cells is possible using Multicolor Flow Cytometry (see figure). Here, we provide a variety of protocols to aid in the preparation of cells for flow cytometry (how to do a proper antibody titration, general procedures to stain extracellular as well as intracellular antigens, etc.).

  • Erythorocyte lysis: From spleen and peripheral blood. May be applicable to different tissues.
  • Antibody Titration: Do not rely on manufacturer specifications, since they may not be necessarily the same as yours!
  • Compensation in Flow Cytometry: When the emission spectra of two or more fluorochromes overlap, the fluorescence from those fluorochromes may be detected on a given photomultiplier (photodetector). To correct for this spectral overlap a process of fluorescence compensation is used.
  • General Surface Staining.
  • (Fluorophoro) Conjugation of Monoclonal Antibodies (M. Roederer)
  • Intracellular Staining: A general procedure to stain intracellular antigens. Remember that localization of the intracellular antigen of your interest will determine the protocol to follow.
  • Build your own panel using FluoroFinder webtool: Look for the configuration of the instrument you want to use in our facility (step 1), set the markers you want to use (step 2), and match the markers to available colors using digital worksheet (step 3) and start building up your multicolor panel!

Quick links

Mario Roederer/compensation: papers, website.

Three Rules for Compensation Controls (from The Daily Dongle).