Constructing multicolor panels for flow cytometry can be a challenging task. Follow these guidelines and recommendations to achieve optimized panels.
- Know your instrument and the fluorophores that you can use. To find more information, check your cytometer manual, view our Instrument Guide page, or check with your local flow core facility.
- Use the Brightness Index, which is our relative indicator of fluorescence intensity above the background for each fluorophore, 1=dim, 5=brightest. On the panel constructor tool, we indicate this in parentheses next to each fluorophore.
- When selecting antibodies follow these general rules:
- Choose the brightest fluorophores that can be used on your instrument. In our Brightness Index chart, the brightest fluorophores are Brilliant Violet 421™, PE, and APC, so these should considered first.
- Choose the brightest fluorophore for your least expressed protein and the dimmest fluorophore for your most highly expressed protein. To help you, we have compiled a chart to indicate the expression of common surface molecules on blood cells. Generally, molecules like CD45, CD3, CD4, and CD8 are quite highly expressed on their target cell types and can be used quite successfully with dim fluorophores.
- Choose fluorophores with emissions having the least spectral overlap. For example, although Brilliant Violet 421™ and Pacific Blue™ do not have exact the same emission spectra, they did have significant overlap, so you should generally avoid using these together. For each laser, you can preview the emission spectra for the available fluorophores using the Emission button to the right of the panel selector. You can also use our Spectra Analyzer to view excitation and emission spectra.
- Use tandems (PE/Cy5, PE/Cy7, APC/Cy7) with caution, as they are more susceptible to degradation by light exposure or fixation. They are essential to large multicolor panels, so just use them with care to prevent light exposure and use appropriate fixation buffers and protocols.
- PE/Fire™ 780 is designed for ASR use only. For more information, check out our ASR page.
- When constructing the panel, choose your specificities with the fewest format options first and specificities with the most options last.