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Get all the key points about how to optimize a flow cytometry experiment, from experiment design, sample preparation, fluorescence staining, detection to data analysis.
In flow cytometry, laser light is usually used to excite the fluorochromes. These lasers produce light in the UV and/or visible range. Fluorochromes are selected based on their abilities to fluoresce with the wavelengths of light produced by the lasers. Therefore, if a flow cytometer has only one laser that produces only 488 nm light, then only fluorochromes that are excited by 488 nm light can be used. The chemical properties of the fluorochrome determine whether its electrons can be excited to the higher energy state by a specific wavelength of laser light. If the electrons can be excited to the higher energy state, the chemical properties of the fluorochrome will also determine the amount of energy lost as heat when the electrons drop back down to the lowest singlet excited state and the wavelength of light produced when the electrons return to their ground state.
The electrons of a fluorochrome can be excited by a range of wavelengths of light. For example, the fluorochrome, fluorescein, will fluoresce when hit by light with a wavelength between 430 nm and 520 nm. However, the closer the excitation wavelength is to 495 nm, the more fluorescence will be produced. This optimal wavelength is called the excitation peak. Similarly, the light produced by fluorochromes has a range of wavelengths. The emission of light from fluorescein, ranges from 490 nm to 630 nm, and the emission peak is approximately 520 nm.
Knowing the excitation and emission properties of fluorescent compounds makes it possible to select combinations of fluorochromes that will work together optimally on a specific flow cytometer with specific lasers. However, for a fluorochrome to be useful in a biological application, it must attach to or be contained within a particle of biological significance. Some fluorochromes are useful because they bind to specific chemical structures, such as antibodies or the nucleic acids in DNA or RNA.
Fluorochromes that are used most often in flow cytometry are ones that attach in some way to biologically significant molecules and are excitable by the lasers that are commonly found on commercial flow cytometers. Many fluorochromes can be attached to antibodies, which will then bind to specific chemical structures on or inside of cells. If these chemical structures are unique to a specific type of cell, then the fluorochrome will identify that cell type. This technique of identifying cells using fluorescent antibodies is called immunophenotyping.
A list of the fluorochromes used most often in immunophenotyping is shown in Table 1 with their peak excitation and emission wavelengths and the laser wavelengths most often used to excite them on a flow cytometer. Table 2 shows the lasers that can generate the required wavelengths of light to excite the various fluorochromes. Some other common applications of fluorochromes in flow cytometry include the detection of intracellular calcium, measurement of the relative amount of cellular DNA or RNA, and measurement of transcription levels using a fluorescent protein as a reporter gene. Fluorochromes used for these applications are shown in Table 4.
|Fluorochrome||Excitation Peak (nm)||Emission Peak (nm)||Laser Wavelengths (nm)|
|Alexa||350||350||445, 334-364, 351-356|
|Marina Blue||365||460||334-364, 405, 407|
|Cascade Blue||395||420||405, 407|
|Cascade Yellow||400||550||405, 407|
|Pacific Blue||405||455||405, 407|
|Phycoerythrin (PE)||565||575||488, 514, 568|
|PE-Texas Red||565||615||488, 514|
|Alexa 633||630||650||633, 635, 647|
|Alexa 647||647||670||633, 635, 647|
|Allophycocyanin (APC)||650||660||633, 635, 647|
|Cy5||650||665||633, 635, 647|
|APC-Cy7||650||770||633, 635, 647|
|Alexa 660||660||690||633, 635, 647|
|Cy5.5||675||695||633, 635, 647|
|Alexa 680||680||700||633, 635, 647|
|Alexa 700||700||720||633, 635, 647|
|Solid-State Violet Laser||405|
|Laser||Channel||Filter Info||Common Fluorophores||Alternate Fluorophores|
|Calcium||FL-1||515-545nm||FITC||Alexa 488, GFP, YFP|
|Calcium||FL-3||670LP||PerCP||PE-Alexa 700, PE-Cy5.5|
|Calcium||FL-4||653-669nm||APC||Cy5-Alexa 647, TOTO-3|
|Application||Fluorochrome||Excitation Peak (nm)||Emission Peak (nm)||Laser Wavelengths (nm)|
|Calcium||Indo-1 (calcium)||325||400||334-364, 351-356|
|Calcium||Indo-1 (nocalcium)||345||485||334-364, 351-357|
|Calcium||Fura Red||485||675||458, 488|
|DNA Content||Hoechst 33342||355||455||334-364, 351-356|
|DNA Content||DAPI||360||460||334-364, 351-356, 405, 407|
|DNA Content||Acridine Orange||495||535||488|
|DNA Content||Propidium Iodide||305||620||334-364, 351-356|
|535||620||488, 514, 568, 633, 647|
|DNA Content||7-AAD||545||650||488, 514, 568|
|DNA Content||To-Pro-3||640||655||633, 635, 647|
|Reporter Gene||Ds-Red||555||585||514, 568|
1. Haugland RP. Handbook of fluorescent probes and research products. 9th ed. Eugene, OR: Molecular Probes; 2002.
2. Shapiro HM. Practical flow cytometry. 4th ed. New York: Wiley-Liss; 2003.