Flow cytometry protocols handbook
Protocols that fit your needs in flow cytometry ranging from sample preparation to numerous cell stimulation conditions, staining, immunophenotyping, and data analysis strategies.
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| 5 | 633/647 | 660/20 | 647 | 665 | (in buffer) 3 (in antifade) 5 | microscopy; flow cytometry; fluorescence microplate reader |
Invitrogen Cyanine5 (Cy5) dye is a bright, far-red-fluorescent dye with excitation designed for use with the 633 nm or 647 nm laser lines. A significant advantage to using long wavelength dyes such as Cy5 dye or Invitrogen Alexa Fluor 647 dye over other fluorophores is the low autofluorescence of biological specimens in this region of the spectrum. The Cy5 fluorophore is also the basis for the lipophilic neuronal and long-term Dil cell tracing reagents, which append alkyl tails (≥12 carbons) onto the core fluorophore.
For stable signal generation, Alexa Fluor 647 dye is generally brighter than Cy5 dye in protein and nucleic acid conjugates. Allophycocyanin (APC) is another bright fluorophore that is spectrally equivalent to Cy5 dye and Alexa Fluor 647 dye, but it is used almost exclusively in flow cytometry.
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Figure 1. Zebra fish brain sample stained with green and orange CellTracker CM-Dil stains. A 30 µm–thick section of a zebrafish embryo stained with CellTracker CM-DiI prior to immunohistochemical analysis. A one-day-old zebrafish embryo was immobilized and impaled in the hindbrain with a microelectrode filled with 1 mg/mL CellTracker CM-DiI in 95% ethanol. One day later, the brain was fixed, embedded, frozen in liquid nitrogen and sectioned on a cryostat. After blocking with 0.1% Triton X-100 in PBS containing 2% BSA and 2% normal goat serum, the 30 µm–thick section was incubated with primary anti-glial antibody in conjunction with fluorescein goat anti–mouse IgG antibody. This section was then viewed sequentially through optical filter sets appropriate for rhodamine and fluorescein, and the resulting images were superimposed. The image was contributed by William Trevarrow, Beckman Institute, California Institute of Technology.
Figure 2. Comparison of the brightness of Alexa Fluor 647 and Cy5 dye antibody conjugates (prepared in our laboratories). More Alexa Fluor 647 dye molecules can be attached to proteins and nucleic acids without significant quenching, thus yielding conjugates that are much brighter than those possible using the Cy5 dye.
Additional resources
Fluorophore and reagent selection guide for flow cytometry
A handy reference poster featuring the broad range of our available dyes and labeling reagents.
Imaging protocol handbook
Protocols that fit your needs in imaging ranging from sample and assay preparation to staining, labeling, and data analysis strategies.
Fluorophore and reagent selection guide for cell imaging
A tool for selecting the optimal fluorescent dyes for your Invitrogen EVOS cell imaging systems.
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