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.
- Basic concepts in compensation: a quick overview (from Mario Roeder's website)
- Fluorescence Minus One (FMO) vs Isotype Controls: In addition to compensation controls, there are several other controls that can, and in most cases, should be used to help resolve issues in staining. Fluorescence Minus One (FMO) controls can help identify gating boundaries, isotype controls can help identify staining issues, and unstained controls show you the background or autofluorescence of the system.
- Compensation Controls: How prepare compensation controls to correct for spillover.
- 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!