Cancer Institute

  • Selecting Fluorochromes for Analysis and Sorting

    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 configuration

    MoFlo filter configuration*

    LaserFilterPMT Laser

    Filter

    PMT
    488nm525/40
    575/30 
    1
    488nm529/28
    575/25
    1
    2
     620/30
    695/30
    3
    4
     625/26
    670/30 
    3
    4
     755LP5355nm405/305
    638nm660/206635nm720/136

    *The filter configuration on the MoFlo is interchangeable.

    • To obtain optimal results, use 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. See spectral viewer:
    • 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.

    Some of the most commonly used fluorochromes and their excitation, emission wavelengths and other properties are listed below:

    • FITC (Ex-Max 494 nm/Em-Max 520 nm): Fluorescein isothiocyanate has a very high efficiency of energy transfer from absorbed to emitted light and is one of the most commonly used fluorochromes. However, FITC is highly sensitive to pH changes and photo bleaching. In addition FITC is relatively dim and should be reserved for highly expressed markers whenever possible.
    • Alexa Fluor® 488 (Ex-Max 495 nm/Em-Max 519 nm): It has nearly identical emission and excitation maxima as FITC. However, Alexa Flour® 488 display higher photostability and is less susceptible to self-quenching. In addition, Alexa Flour® 488 is pH insensitive (over a broad pH range) and tends to be brighter compared to FITC on most instruments. These properties, i.e. increased sensitivity and environmental stability, make Alexa Flour® 488 suitable for intracellular staining. Due to nearly identical excitation and emission properties FITC and Alexa Fluor® 488 cannot be used simultaneously.
    • CFSE (Ex-Max 494 nm/Em-Max 521nm): Carboxyfluorescein Diacetate is a cell permeable dye most commonly used to track cell division and proliferation. CFSE enters the cell via diffusion and is cleaved by intracellular esterases, generating a fluorescent product that covalently binds to primary amines. CFSE remains stabile in non-dividing cells for several days and upon cell division the dye is equally distributed into the two daughter cells. This results in a sequential reduction of the initial fluorescence proportional to the number of divisions.
    • PE (Ex-Max 496 nm/Em-Max 578 nm): R-phycoerythrin is a pigment naturally found in red algae where it transfers light energy to chlorophyll during photosynthesis. PE has one of the largest absorption co-efficiencies making it one of the brightest fluorochromes available to date.
    • PE-Texas Red® (Ex-Max 496 nm/Em-Max 615 nm): It is a tandem conjugate that combines PE and Texas Red®. Like other tandem conjugates, it is a combination of two fluorochromes where the excited fluorochrome is able to transfer its fluorescent energy to the second molecule, which then fluoresces at a different, longer wavelength. Basically, light emitted from the first fluorochrome (PE) excites the second (Texas Red), which then fluoresces at a 615 nm maxima. Special care must be taken when using PE-Texas Red® conjugates in conjunction with PE since there is considerable spectral overlap in the emission profiles of both fluorochromes.
    • PI (Ex-Max 536 nm/Em-Max 617 nm, when bound to nucleic acid): Propidum iodide is an intercalating agent that binds non-specifically to nucleic acid with the stoichometry of 1 dye per 4-5 bases. Because PI non-specifically binds to all nucleic acids it is necessary to treat the cells with nucleases to distinguish between DNA and RNA. Once PI is bound to the nucleic acid its fluorescence is enhanced 20-30 fold. PI is membrane impermeable and generally excluded from viable cells. PI is commonly used to stain DNA for cell cycle analysis and the cells must be permeabilized prior to incubation with the dye. PI is also commonly used as a cell viability marker, as it is excluded from healthy cells, but easily penetrates the disrupted membranes of dead/dying cells. PI is a potential mutagen and should be handled with care.
    • PE-Cy™5 (Ex-Max 496 nm/Em-Max 667 nm): It is a tandem conjugate that combines phycoerythrin and a cyanine dye. Because of its broad absorption range and the fact that its emission spectra is equivalent to APC, PE-Cy5 is not recommended for simultaneous use with APC. The cyanine dyes are known to exhibit non-specific binding to Fc-receptors, which is most apparent on monocyte populations.
    • PerCP (Ex-Max 482 nm/Em-Max 678 nm): PerCP proteins are carotenoid-protein complexes derived from phytoplankton. Due to its photobleaching characteristics, PerCP conjugates are not recommended for use on flow cytometers with high-power lasers (>25 mW), such as the MoFlo XDP.
    • PerCP-Cy™5.5 (Ex-Max 482 nm/Em-Max 695 nm): It  is a tandem conjugate that combines PerCP with a cyanine dye. PerCP-Cy5.5 is not subject to photobeaching like PerCP and can be used with stream-in-air flow cytometers. In addition, PerCP-Cy5.5 displays less Fc-receptor mediated nonspecific staining than PE-Cy5. Additionally, the PerCP-Cy5.5 tandem conjugate is not as susceptible to fixative or light instability as compared to APC-Cy7 and PE-Cy7.
    • PE-Cy™7 (Ex-Max 496 nm/Em-Max 785 nm): It is a tandem fluorochrome that combines PE and a cyanine dye. PE-Cy7 conjugated reagents are as bright as PE conjugates. However, PE-Cy7 is highly sensitive to photo-induced degradation resulting in loss of fluorescence. Care should be taken to avoid light exposure and prolonged exposure to paraformaldehyde fixative. Fixed cells should be analyzed within 4 hours of fixation in paraformaldehyde or transferred to a paraformaldehyde-free buffer for overnight storage.
    • APC (Ex-Max 650 nm/Em-Max 660 nm): Allophycocyanin is an accessory photosynthetic pigment found in blue green algae. APC has six phycocyanobilin chromophores per molecule, which make it a very bright fluorochrome that is highly suitable for flow cytometry applications. APC is excited by the red diode laser and excites in several tandem dyes including APC-Cy5.5 and APC-Cy7.
    • Alexa Fluor® 647 (Ex-Max 650 nm/Em-Max 668 nm): Conjugates are highly photostable and remain fluorescent over a broad pH range. The excitation and emission maxima are nearly identical to those of APC, albeit APC tends to be brighter. However, Alexa Fluor® 647 is considered more optimal for intracellular applications. Due to nearly identical excitation and emission properties, but different spillover characteristics, APC and Alexa Fluor® 647 cannot be used simultaneously.