This month we explore the ever-expanding selection of commercially available fluorescent antibodies and how researchers are getting better data by designing more colorful panels.
Sections:
- Research Trend Toward More Colors
- Suppliers Expanding Fluorescent Catalogs
- Simplifying Your Selections
The size and complexity of flow cytometry experiments have expanded rapidly over the past few years. Cytometers can now simultaneously analyze up to 30 different fluorochromes, and new data analysis software can handle far more information than ever before. Researchers who typically ran three to four color panels just a few years ago are now designing experiments with twice as many colors. This increases the number of measurable parameters, provides more data, and generally enlarges the overall scope of the study.
Every Color in the Rainbow
To keep up with this trend, reagent suppliers have greatly expanded their fluorochrome-conjugated antibody catalogs. FluoroFinder’s reagent database now contains over 550 fluorochromes (from 45 suppliers) for use in multi-color flow cytometry, microscopy and more. Here is a brief overview of our current fluorochrome database, from the tried and true (FITC, PE, APC) to the new unique and proprietary dyes.
Low MW Compounds The first fluorophores available were low molecular weight compounds. These include fluorescein (FITC), rhodamine (TRITC), coumarin, and cyanines. High MW Compounds Relatively higher molecular weight compounds isolated from cyanobacterial or algal sources include phycoerythrin (PE) and phycocyanin. Together with the low MW compounds, these fluorophores are very well characterized and widely available. Tandem Dyes Tandems are composed of two covalently attached fluorescent molecules, where one serves as the donor and the other as acceptor. They behave as unique fluorophores with the excitation properties of the donor and the emission properties of the acceptor. These dyes are considered ideal for multiplexing experiments, as researchers can obtain several readouts from a single sample. Examples of tandem dyes include PE/Cy5 and APC/Cy7. Quantum Dots Quantum dots (Qdots) are very small semiconductor particles, typically only ~2-10 nanometers in size. Qdot exciton lasts much longer than that of traditional fluorescent fluorophores, making them ideal for low-density antigen detection. Fluorescent Proteins Used in a wide variety of cellular biology applications, fluorescent proteins including GFP, YFP, RFP, etc. are often useful for flow cytometry analysis. Check out our video on Tracking Your Fluorescent Proteins with FluoroFinder. Proprietary Dyes Many reagent suppliers have developed their own proprietary dyes families. While they may be similar to other commercially available options, they typically have unique spectra curves to consider when planning an experiment. Examples of proprietary dye families include.
| Dye Family | Supplier |
|---|---|
| Alexa Fluor® | Thermo Fisher/Invitrogen/Molecular Probes |
| Atto Dyes® | Thermo Fisher/Sigma Aldrich |
| Brilliant™ Violet/UV | BioLegend/BD Biosciences |
| CF™ Dyes | Biotium |
| DY Dyes | Dyomics |
| DyLight® Fluor | Thermo Fisher |
| eFluor® Dyes | Thermo Fisher/eBioscience |
| HiLyte™ Fluor | Anaspec |
| Super Bright | Thermo Fisher |
| Vio® Dyes | Miltenyi Biotech |
More Colors, More Problems?
This plethora of color options can easily overwhelm researchers during experiment design. Researchers search multiple suppliers to find the right combination of antibodies and fluorochromes for their experiments. All too often they simply return to the same fluorochromes they have used in the past, despite new brighter options being available. FluoroFinder seeks to address this issue by 1) restricting options based on your specific cytometer’s laser/filter configurations and 2) showing all commercially available options in one, easy to compare, location. This allows researchers to build panels efficiently; being introduced to new antibody/fluorophore combinations that fit their specific needs, without being overwhelmed by options that won’t. Don’t be afraid to experiment with more/new colors! Expand your experimental palette by building your next panel on FluoroFinder.
