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Flow cytometry is a powerful tool for identifying and analyzing multiple antigens simultaneously. However, increasing the number of antigens and fluorochromes also increases the complexity of the experimental design. Overcome these challenges by following these five steps when designing your panel:
Before you start designing your multicolor flow panel, find out (a) the number and type of lasers, (b) the number of detectors, and (c) the type of filters that are available on your flow cytometer. These components will determine the constraints on which fluorochromes you can use.
Match the excitation wavelength of your fluorochromes to the lasers available. Furthermore, match the emission wavelength of your fluorochromes to the available filters and which wavelengths they allow to pass.
Refer to your instrument’s manual or speak to your core facility manager to ensure optimal detection.
Some cell populations are rare or the antigen density is low due to functional differences and cell activation levels. As a general rule, use the brightest fluorochromes, such as PE for targets of low or unknown antigen expression and/or rare cell populations. Targets with high antigen expression can be matched with dimmer fluorochromes, such as PerCP. It is important to note that fluorochrome brightness can also differ due to multiple factors such as the buffers used and type of flow cytometry used.
The relative brightness of your fluorochromes can be checked using our fluorochrome chart.
Selecting fluorochromes with little or no spectral overlap can be conflicting when trying to select the brightest fluorochrome. However, it is worth sacrificing some brightness in one detector to avoid spillover. By ensuring there is minimum overlap, it will decrease the amount of compensation needed.
Controls including unstained cells, live/dead markers, single-staining positive controls, and fluorescence-minus-one-staining are vital for complex multicolor panels. See our recommended controls guide for more information.
Antibody concentration: non-optimal concentrations of antibody can increase non-specific binding or reduce the sensitivity of the measurement. Therefore, all antibodies should be titrated to determine the best signal-to-noise ratio.
Fc blocking: phagocytic cells (such as monocytes) have Fc receptors (FcR’s) on their surface, which can non-specifically bind to the Fc region of antibodies (but not as a receptor-ligand interaction). This binding can be blocked with the addition of FcR blocking reagents prior to staining, such as an anti-CD16+CD32 antibody (ab25235) for mouse samples or human IgG in human samples. This blocking step should be included in homogenized tissue samples, which may contain macrophages, as well as cell culture lines such as Daudi and THP-1.
By following these five important steps, you’ll be able to design and run complex multicolor flow cytometry experiments while avoiding many of the common pitfalls. For more information on flow cytometry, visit our resource page: