Designing multicolor panels for flow cytometry analysis is complicated and tricky. Here are ten handy tips for a successful multicolor flow cytometry experiment.


Familiarize yourself with your flow cytometer

It is important to understand the capabilities of your flow cytometer. Most cytometers have two or more lasers selected from five wavelengths: ultraviolet (355 nm), violet (405 nm), blue (488 nm), yellow (561 nm), and red (640 nm). In addition to the lasers, specific filters and detectors determine the configuration. These components define how many colors can be detected simultaneously (for new cytometers this can be more than 17). Regular cleaning and calibration using fluorescent beads will allow you to get the most out of your flow cytometer.


Prepare your sample correctly

Poorly prepared or unhealthy cells can lead to suboptimal results due to cell death or clumping. It is also important to treat the cells gently and avoid vortexing when making cell suspensions. During staining, cells should be kept on ice if the experiment permits. To reduce the formation of aggregates, which can block the flow cell, high cell densities should be avoided. Adding DNAseI or EDTA as well as filtering can also help to reduce aggregates.


Remove the dead cells

Dead cells can lead to false positives due to autofluorescence and increased non-specific antibody binding. Dead cells cannot always be removed by forward scatter (FSC) and side scatter (SSC) gating, so it is best to use a live/dead exclusion marker. Propidium Iodide (ReadiDrop™ Propidium Iodide) or 7-AAD (ReadiDrop™ 7-AAD) are commonly used to identify the dead cells, but they cannot be used with fixed cells. Fortunately, there is a range of VivaFix™ live/dead cell reagents that can be used with live or fixed cells.


Optimize your staining protocol

Nonspecific binding of antibodies due to charge or protein-protein interactions can lead to false positives. Such nonspecific binding can be reduced by including BSA or unconjugated antibody in your staining buffer to block these interactions. Furthermore, many cells (for example, B cells, NK cells, and macrophages) have Fcg receptors on their cell surfaces, which can bind to the Fc region of your antibody and result in nonspecific staining. These can be blocked by the addition of FcR blocking reagents or the use of F(ab)2 fragments that do not contain an Fcg portion. High antibody concentrations can also increase nonspecific binding. Therefore all antibodies should be properly titrated to maximize the signal-to-noise ratio. Using appropriate antibody concentrations is critical for multicolor flow.


Decide if and when isotype controls are to be included

You may not wish to use isotype controls (Herzenberg 2006, Maecker and Trotter 2006). But if you do, make sure you have included other appropriate controls (see tip 6). Isotype controls have been developed for surface staining to determine what is specific. The correct isotype control is an antibody generated against an irrelevant antigen (for example, KLH) of the same antibody subclass, with the same conjugated fluorophore, that is obtained from the same supplier as your test antibody and used at the same concentration. Its purpose is to ensure that observed staining is due to specific antibody binding rather than an artefact, to exclude nonspecific binding to Fcg receptors, and to exclude nonspecific binding of the antibody or the fluorophore (such as PE) to cellular components.


Be prepared for problems with intracellular staining controls, permeabilization, and nonspecific binding of conjugates

Performing intracellular staining can be more challenging than cell surface staining. In this case, isotype controls may not be appropriate. It is good practice to have several controls to more accurately determine your positive population. These can include an unstained sample, a negative sample stained with antibody that does not express the antigen of interest, or a known positive sample. There are several permeabilization protocols that use commercial reagents (for instance, Leucoperm™) alone, in conjunction with methanol, or with mild detergents such as saponin. Different methods might improve the staining, but some conjugates need to be avoided (for example PE when using methanol). Finally, avoid using biotin and FITC in intracellular panels if possible. Endogenous biotin has to be blocked with unconjugated streptavidin and FITC can bind nonspecifically through electrostatic interactions.


Build effective multicolor panels

When the emission spectra of two fluorophores overlap, spillover of one fluorophore into the detection channel of the other might be observed (for example, FITC and PE). This can make it difficult to identify discrete populations without the use of spillover correction by a technique called compensation. Where possible, the spillover can be minimized by selecting fluorophores that have little or no overlap. But this is not always possible when using several fluorophores. A spectrum viewer can assist in obtaining the best fit emission profiles. Using fluorophores with little or no overlap for cell subset markers, such as T cell subsets, and closely overlapping fluorophores for mutually exclusive markers, such as CD3 and CD19, can prevent reduced sensitivity due to fluorescence spread.


Prepare appropriate controls for multicolor panels

With multicolor panels, single-stained compensation controls are essential for calculating compensation values. Compensation values can also be determined using antibody binding beads labeled with your proposed multicolor panel. Finally, fluorescence minus one (FMO) controls can determine fluorescence spread and gating boundaries and can help avoid reduced sensitivity.


Know your biology

Fluorophores with the brightest stain index (such as PE) are best used for cells with the lowest antigen expression or the smallest subset, whereas dimmer fluorophores are more suitable for more highly expressed antigens. However, when using several colors, you may have to make compromises, depending on availability of fluorophores and antibodies and flow cytometer configuration. Tandem dyes can complicate the process due to the spillover fluorescence of the donor fluorophore into other fluorophore channels. Staining index tables to minimize this are available to assist you with multicolor panel design.


Treat your fluorphores with care

As you increase the number of colors in your panel, you will have to use tandem dyes such as PE-Alexa-Fluor 647 or APC-Cy7, which increase the emission range from a single laser. However, they are prone to degradation by light and cellular biological activity, which will result in uncoupling and fluorescence emission at two wavelengths, leading to false positives. The brightness of tandem dyes may be reduced by fixation and permeabilization, so ideally these steps should be as mild as possible. Compensation values should be obtained from samples treated in an identical manner to account for this.


Herzenberg LA et al. (2006). Interpreting flow cytometry data: a guide for the perplexed. Nat Immunol 7, 681–685.

Maecker HT and Trotter J (2006). Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69, 1037–1042.

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