This article offers an overview aimed at selecting the right multicolor panels to carry out flow cytometry.
Flow cytometry is a platform with the power to carry out the identification and characterization of multiple antigens at one time. At the same time, it must be noted that when more antigens and fluorochromes are employed, the experimental design becomes much more complex. To deal with this task, it is useful to consider the following five steps.
1. Understanding Your Flow Cytometer
The first step in designing the multicolor flow panel is to determine how many and what type of lasers are to be used, the number of detectors to be used, and the filter types that are available on the flow cytometer. These results influence the limits on the fluorochromes that can be used.
The wavelength at which the fluorochromes are excited must then be matched with the available lasers. After this is achieved, the wavelength at which fluorochrome emission occurs is matched to the available filters, as well as to the wavelengths that are allowed to pass.
2. Cell Population, Antigens and Fluorochromes
Some cell populations have a very low density, or the density of the antigen may be very low because of differences in the way that they function or in the levels of cell activation.
In general, the fluorochromes chosen should be the brightest, like PE when the target’s antigen expression is low or the level is not known, as well as when the cell populations are very rare. When the target antigen is expressed at high levels, dimmer fluorochromes may be used, such as PerCP. The brightness of the fluorochrome may vary with many factors, like the type of buffer or flow cytometer used.
3. Spectral Overlap
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 in order to avoid spillover. By ensuring there is as little overlap as possible, it will reduce the amount of compensation needed.
Controls including unstained cells, single-staining positive controls, live/dead markers and fluorescence-minus-one-staining are essential for complex multicolor panels.
5. Staining Optimization
With regard to 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 in order to determine the best signal-to-noise ratio.
In Fc blocking, phagocytic cells like monocytes carry surface Fc receptors (FcR) that are capable of non-specific binding to the Fc region on the antibodies, without a true receptor-ligand interaction. To prevent this type of binding, FcR-blockers must be added as reagents before staining is applied, such as, for instance, an anti-CD16+CD32 antibody (ab25235) that prevents non-specific binding in mouse samples, or human IgG in human cell 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 can design and run complex multicolor flow cytometry experiments while avoiding many of the common pitfalls.
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