For the development of flow cytometry, the most important driving force is antibodies, followed by fluorescent dyes that match the detection capabilities of the instrument. Fluorescent dyes are still not keeping pace with the number of instrument detection channels.
Existing flow-through fluorescent dyes can be divided into the following 11 categories:
1. Small organic molecules
When it comes to small organic molecules, most people must be confused, but if I give a few examples, it will definitely dawn on me.
FITC (389 Da), Alexa Fluor 488 (FITC analog, 643 Da), Texas Red (TxRed, 625 Da), Alexa Fluor 647 (1155 Da), Pacific Blue (242 Da), and Cy5 (242 Da) 762 Da). Is it basically used?
These small organic molecules have a consistent emission spectrum and small Stokes shift (difference between excitation and emission wavelengths, approximately 50-100 nm), are photostable, and are easily conjugated to antibodies, so they are widely used .
It is worth mentioning that Alexa Fluor dyes are more photostable than FITC, so if you want to image, they are the choice.
Phycobiliproteins are large protein molecules extracted from algae such as cyanobacteria and dinoflagellates. For example, phycoerythrin (PE) has a molecular weight of 240,000 Da. These proteins have large Stokes shifts (75-200 nm) and stable emission spectra. Because phycobiliproteins are large, they typically have a 1:1 protein to fluorochrome ratio during conjugation, making them useful for quantitative flow cytometry.
However, phycobiliproteins are susceptible to photobleaching, so prolonged or repeated exposure to excitation light sources is not recommended.
In the phycobiliprotein family, in addition to phycoerythrin (PE), there are allophycocyanin (APC) and Momordica chlorophyll protein (PerCP).
3. Quantum dots
Quantum dots (Qdots) are semiconductor nanocrystals with fluorescence emission spectra that are closely related to the size of the nanocrystals. They are best excited by ultraviolet or violet light, but are also easily excited by other lasers in small amounts, so when using Qdots in multiparameter experiments, this tiny excitation of other wavelengths of lasers complicates fluorescence compensation.
As such, although quantum dot dyes have high brightness, these reagents are mostly replaced by polymer dyes in multiparameter schemes due to the intractability of these compensation issues and the difficulty of binding Qdots to antibodies.
4. Polymer dyes
Macromolecular (polymer) dyes consist of polymer chains that collect light signals and can "tune" the absorption and emission of specific wavelengths of light, depending on the length of the polymer chain and the molecular subunits that are attached. These dyes are very stable and have quantum efficiencies similar to phycobiliproteins with greatly improved photostability.
Since the polymer dyes can be made to absorb only specific wavelengths of light, the problem of cross-wavelength excitation of Qdots is avoided.
Typical representatives of polymer dyes are Brilliant Violet (BV), Brilliant Ultraviolet (BUV) and Brilliant Blue (BB).
5. Tandem dyes
Tandem dyes chemically couple phycobiliproteins (PE, APC, PerCP) or macromolecular dyes (BV421, BUV395) with organic small molecule fluorescent dyes (Cy3, Cy5, Cy7), and use fluorescence energy transfer (FRET) to form a single laser More wavelengths of emitted light are excited.
For example, the excitation maximum of Texas Red is 589 nm, while the emission of PE is 585 nm, so by coupling PE with Texas Red, the emission light of PE excites Texas Red through FRET, thus making PE-TxRed available at 488 nm or 532 nm Laser excitation.
The same method can also be used for polymer antibodies in tandem with organic small molecule dyes.
Tandem dyes are very bright and have large Stokes shift values (150-300 nm), which are useful when detecting low-density antigens. However, tandem dyes are less stable than donor fluorochromes, and energy transfer efficiency varies from batch to batch, complicating compensation.
6. Heavy metal ions
Heavy metal ions are not strictly speaking fluorescein, but we also mention them here.
Antibodies used in mass cytometry are not conjugated with fluorescein, but are conjugated to heavy metal ions of a single isotope in the lanthanide series. There are currently 35 lanthanide isotopes commercially available for antibody conjugation. These probes are non-fluorescent and only suitable for mass cytometry.
7. Fluorescent protein
Fluorescent proteins are frequently used as reporter systems for gene expression. The most commonly used is green fluorescent protein (GFP) from Aequorea victoria. From this, CFP and YFP are derived.
The red fluorescent protein (DsRed) is derived from the mushroom sea anemone Discosoma, and the next-generation monomeric fluorescent proteins (mCherry, mBanana) are derived from DsRed and have broader excitation and emission spectra.
With the maturity of gene cloning technology, there are hundreds of fluorescent proteins, whose excitation and emission spectra range from ultraviolet to near-infrared.
8. Nucleic acid dyes
Nucleic acid dyes bind DNA, RNA, or both. They can be used to quantify DNA for cell cycle analysis (e.g. PI, 7-AAD, DyeCycle Violet, DAPI), sort chromosomes (e.g. Hoescht 33342, Chromomycin A3), sort stem cells using side population analysis (e.g. Hoescht 33342), Live and dead cells are differentiated and bacteria are sorted.
The level of proliferation can be determined by combining nucleic acid dyes with another label, such as a fluorescent dye-conjugated anti-bromodeoxyuridine (BrdU).
9. Cell proliferation dyes
Cell proliferation is measured by incubating cells with BrdU (bromodeoxyuridine), which is incorporated into cellular DNA synthesis, and then stained with antibodies and DNA dyes directed against BrdU. However, this method is not suitable for long-term proliferation studies.
Carboxyfluorescein succinimidyl ester (CFSE) can be used to track multiple divisions of proliferating cells. Red and purple variants of CFSE dyes are also now available. At appropriate concentrations, these dyes do not affect cell growth or morphology, making them suitable for long-term proliferation studies.
10. Cell death dye
Cell viability can be determined by exclusion dyes (eg, propidium iodide, DAPI) or by the binding of dyes to intracellular amines.
Exclusion dyes cannot be fixed and are only suitable for cells that are not infectious and require immediate analysis.
Amine dyes including Live/Dead (ThermoFisher), Zombie (Biolegend) or Fixable Viablity (BD Biosciences) are suitable for fixed cells and therefore those with infectious cells, cells stained with intracellular antigens, cells fixed with paraformaldehyde .
11. Calcium indicator dye
Calcium indicator dyes undergo a color shift upon binding to calcium and can be used to indicate cellular activation and signaling.
The most commonly used dye is still indo-1, the UV biphasic calcium probe. There are also Blue-green calcium probes for fluo-3.