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FACS Core Facility  / Multicolor Experiments

Multicolor Experiments

Before you set up a multicolor experiment it is worthwhile to have a look at the spectrum viewer (Joe Trotter, BD Biosciences), or the fluorochrome color table (David Chambers, Salk institute) and make yourself familiar with the optical properties of the reagents currently available.

The Pharmingen reference site gives you information where the acronyms come from. You then can call us and talk about the panel you want to use, and believe us it is easier when you already know the vocabulary. It gives you also some information if your reagents can be visualized with our instruments (see also Multicolor Technote) and if they can interfere with each other.

The FACSAria cell sorter has 3 built in solid state lasers, with excitations of 407nm, 488nm and 633nm. In combination with the standard 488nm illumination you can also perform coexpression studies on several fluorescent proteins avoiding crosstalk, which you get on a single laser instrument. For further details have a look at the Clontech brochure.

The 407nm laser line present can be used for the analysis of CFP expression in combination with EGFP or EYFP, a setup used for the measurement of Fluorescence Energy Transfer (FRET) for protein interaction studies. It is also possible to perform cell cycle analysis using Hoechst or DAPI in combination with multi-color immunophenotyping

 Controls Required for Multi-color Flow Acquisition

For an introductuary discussion about problems you can run into using multicolor setup take a look at the Mario Roederer compensation tutorial.

Compensation Controls: The new digital instrumentation (LSRII and DIVa's) allows for a complete spillover (compensation) matrix to be computed and applied to the experiment. To properly execute this function, the new instrumentation requires the following:

  • Unstained cells to determine background for compensation computation
  • Positive controls for each color
  • Background (FMOC) Controls if you are interested in real expression levels
  • Isotype controls for a simple experiment

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