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Selecting Fluorochromes for Flow Cytometry

In order to obtain optimal results from the flow cytometry analysis, especially when performing multidimensional analysis, it is vital that the appropriate combination of fluorochromes is used. When choosing fluorochromes, there are a number of different parameters to take into account. Each fluorochrome has distinct properties and is characterized by specific excitation and emission wavelengths. First, the fluorochrome must be excited by the lasers available on the instrument. Second, the emission wave lengths are read by different detectors or photomultiplier tubes and the range of detection is limited by optical filters.

Currently, the Gallios has a five + one configuration with five photomultiplier tubes for detecting light emitted from a 488 nm excitation source and one for detecting light emitted by the 638 nm excitation source. For example the FL-1 photomultiplier tube is preceded by a 525/40 bandpass filter, this allows wavelengths of 525 +/- 20 nm to pass through to the FL-1 photomultiplier tube. Similarly, the FL-2, FL-3 and FL-4 photomultiplier tubes are preceded by specific bandpass filters, while the FL-5 is preceded by a 755 nm long pass filter that will allow any wavelength >755nm through.

A similar configuration is available on the MoFlo XDP cell sorter with four detectors for light emitted by the 488 nm excitation source and one each for light emitted from the 355 nm UV and 635 nm excitation sources. Furthermore, the individual filters on the MoFlo XDP are interchangeable and can be further optimized.

Gallios Filter ConfigurationMoFlo XDP Filter Configuration*
LaserFilterPMTLaserFilterPMT
488nm525/401488nm529/281
575/302575/252
620/303625/263
695/304670/304
755LP5355nm405/306
638n,660/206635nm720/136

*The filter configuration on the MoFlo XDP is interchangeable.

  • To avoid spectral overlap choose, if possible, fluorochromes that have the minimal amount of emission spectral overlap. In general the further apart the different emission maxima of the fluorochromes are, the less spectral overlap will occur.
  • Use the brightest fluorochrome, typically PE or PE based energy transfer conjugates, for the protein that has the lowest expression, and vice versa the dimmest fluorochrome for the most highly expressed protein.
  • Know the specific properties of the fluorochromes you are using. In addition to the excitation/emission spectra each fluorochrome has specific properties and may be unsuitable for your particular experiment. For example the fluorescence of FITC is highly pH dependent and sensitive to acidic conditions, and as such is not suitable to use if your protocol includes acidic buffers.
  • Avoid exposing samples to bright light as many fluorochromes are light sensitive and subject to photo bleaching.
  • Tandem conjugates are dyes that combine two fluorochromes. For example, the tandem dye PE-Cy5 consists of phycoerythrin and a cyanine dye. In tandem dyes one of the fluorochromes is excited by the laser and is, in turn, able to transfer its fluorescent energy to the second molecule which then emits light of a different, longer wavelength. In many cases the energy transfer is incomplete and depending on the specific tandem dye used, varying levels of compensation are required.