A practical approach to multicolor flow cytometry for immunophenotyping
Introduction
Since the earliest application of flow cytometry to the study of cells, there has been a drive to increase the number of distinct measurements for each cell. This developmental effort blossomed in the late 1990s, when the number of independently measurable ‘colors’ (each color corresponds to a distinct fluorescence-based measurement of a cellular protein or function) increased from five to 11 Roederer et al., 1997, Bigos et al., 1999.
The success of this developmental effort was due to the coordinated development of new hardware, new fluorochromes, and new software analysis tools that significantly increase the quality and quantity of measurements. This increase comes with a price, however, as this new technology has its own set of technical problems and difficulties that users must be aware of and must overcome in order to derive meaningful results. Nonetheless, once these hurdles are overcome, this new technology is well worth the effort, as the information obtained from the measurements is not only novel but could not be obtained otherwise using standard flow cytometric techniques.
As we outline below, setting up multicolor flow cytometry is not simply achieved by adding new reagents to existing reagent combinations, but requires a more involved process of quality control, optimization, and ‘debugging’. Therefore, to distinguish multicolor flow cytometry with its unique benefits and technical problems from current standard flow cytometric technologies (i.e., using four or fewer fluorescent dyes), we refer to it as ‘polychromatic flow cytometry’ (PFC) and will use the term throughout this manuscript.
Flow cytometers capable of collecting data for more than three or four colors are now becoming more prevalent, as manufacturers have recognized the significant demand for the types of analysis afforded by this technology. Given the bewildering array of fluorochromes, lasers, hardware, and software that might be used in PFC, we outline here the fundamental requirements, interactions, and problems associated with setting up this technology, so that users can make educated decisions about instrument requirements and the design of their experiments. Finally, we provide some examples in which this technology has been of particular benefit. We hope to provide with this brief review some practical tips and encouragement for those thinking of expanding their current flow cytometric measurements. The benefits of true multicolor flow cytometry make this technique a particularly useful and probably soon an irreplaceable tool for the study of cell biology and immunology.
Section snippets
Fluorochromes
The ability to measure multiple parameters of each cell is limited by the number of fluorochromes that can be simultaneously measured. The 11-color PFC that is currently in routine use at Stanford uses dyes excited by three different laser lines. The excitation and emission spectra of these dyes and the filters that were chosen to collect the emitted light from these dyes are shown in Fig. 1.
Lasers
One of the single largest component costs of cytometry instrumentation is the cost of the lasers. Newer diode lasers are becoming prevalent and these can be significantly cheaper than the older gas ion lasers. When considering a solid-state laser, it is important to choose one that has a long life span and provides a consistent power output, which therefore excludes the very cheap solid-state lasers that are currently available. In addition, for current stream-in-air instrumentation it is
What is compensation?
Compensation is the process by which the spectral overlap between different fluorochromes is mathematically eliminated. The algorithm of compensation is a straightforward application of linear algebra, and should not be thought of as a subtraction process. Compensation between detectors can be performed either by hardware, after signal detection but before logarithmic conversion and/or digitization, or post-collection by software.
While compensation is one of the most important processes
Choosing the right fluorochrome
As outlined above, fluorochromes differ considerably in relative ‘brightness’. Conjugation of the various fluorochromes to the same antibody and staining of the same population of cells can therefore result in large differences in resolution of positive and negative events (Fig. 2). For single-color staining for a highly expressed marker such as CD8 on T cells (Fig. 2), the differences are merely cosmetic and no alterations in the frequency of negative and positive events are found after
Choosing the right combinations of fluorochromes
What are the criteria for choosing the right combination of fluorochromes? As outlined above, one major criterion is the expected levels of expression for the markers of interest. Markers such as CD5 on B cells can only be identified when a bright conjugate is used; but identifying CD5 on T cells can be accomplished with any fluorochrome. An unknown surface molecule is initially best studied with a conjugate that uses one of the ‘brightest’ fluorochromes, PE, Cy5PE, or APC. Other considerations
Enhanced accuracy of measurement with multicolor flow cytometry
Despite the relatively good resolution that the use of SA-PE, Cy5PE and APC afforded for the staining of CD5 on a small B cell subpopulation in the spleen (Fig. 3), it would be difficult to accurately determine the frequency of these cells using this two-color stain. In Fig. 6 we outline how the simultaneous use of four (Fig. 6B) and six (Fig. 6C) colors enhances the accuracy of the measurement and the amount of information that can be obtained from one stain (Fig. 6A). In this figure we
Staining controls
Control stains are important for all experiments using flow cytometry, but they become particularly critical for complex multicolor stains. The higher the number of fluorochromes and antibodies used in each stain the greater the risk for artifacts introduced by compensation errors and/or reagent interactions. In general, two types of controls should be included and data collected with every experiment: compensation controls and staining controls.
It will become apparent from the discussion below
Advantages of PFC
Most of the advantages of increasing the number of colors in flow cytometry experiments are readily apparent. First, the information content increases geometrically with the number of parameters simultaneously analyzed. Second, and just as important, information can be obtained from multicolor experiments that is not available in any other way. For example, no combination of one-color stains can accurately enumerate or be used to isolate CD3+CD4+CD8− T cells (excluding, for example, CD3+CD4+CD8+
Acknowledgements
The development of multicolor flow cytometry spanned more than half a decade and represents the efforts of a large number of individuals. We thank in particular Drs. Leonard and Leonore Herzenberg in whose laboratory both fluorescence-activated flow cytometry and Polychromatic Flow Cytometry were born. The work, continuous drive and implementation of technical improvements by Dr. David Parks, Marty Bigos, Richard Stovel, Tom Nozaki, Wayne Moore, and Adam Treister have led to the development of
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Present address: Center for Comparative Medicine, University of California, Davis, CA 95616, USA.