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In flow cytometry, fluorescent probes can be used in two different ways: as reporters and as encoders. In reporting applications, the fluorophore serves as a label with which an analytical measurement is made, and examples include fluorescently labeled antibodies, genetically produced fluorescent protein fusions, and fluorescent ion indicators, nucleic acid stains, and lipid probes. Highly multiparameter assays that combine many reporters are a hallmark of modern flow cytometry.

In encoding applications [1], fluorescence is used to convey information about the identity or history of particles in a sample. The classic encoding application involves microspheres encoded with different amounts of one or more fluorophores, such that they can be distinguished based on their fluorescence intensities. Distinct bead sets are functionalized with specific reagents, for example capture antibodies, exposed to sample, and then stained with a fluorescent reporter. After flow cytometry, the signals from the encoding fluorophores are used to identify the bead, while the reporter fluorescence provides the analytical measurement for that bead.

This optical barcoding concept can also be applied to cell analysis for high throughput applications. In cellular barcoding [2], cells undergo experimental treatment in microwell plates, and then cells in each well are fixed and stained with a combination of fluorophores that constitute a unique barcode for each well. Following barcoding, cells from all of the wells are pooled and stained with reporter probes in a single tube, and then analyzed by flow cytometry. The fluorochromes used for barcoding are analyzed to identify which well each cell came from, and the reporter probes provide the analytical measurements for each sample. Cell barcoding can greatly reduce reagent use compared to staining each sample in its individual well, allowing for more cost-effective execution of ambitious experimental designs, such as these that include time courses and dose response measurements [3]. Cell barcoding can also increase assay throughput, as an entire multiwall microplate can, in principle, be analyzed as a single sample on the flow cytometer.

In this issue, Smurthwaite and colleagues (page 105) describe another approach to cell barcoding, one that employs genetically encoded fluorescent protein fusions to barcode cells. The authors developed a collection of stably transfected cell lines expressing one or more fluorescent protein fusions, and sorted single cells to establish clonal lines with discrete levels of fluorescence intensity such that they can be distinguished upon analysis by flow cytometry. They show that the fluorescent protein barcode is stable, and that the cells can be used with other fluorescent reporter probes to perform multiplexed single cell assays. The use of fluorescent proteins to barcode provides a path to performing multiplexed assays on live cells, opening the door to a range of cell functional and kinetic measurements that are not possible or practical with fixed cells.

Multiplexing has emerged as a major application area in flow cytometry, with numerous commercial multiplex assay kits available that use fluorescence intensity-based encoding, and a number of other encoding strategies demonstrated in the literature. One limitation of fluorescence-based encoding is that fluorescence channels dedicated to encoding cannot be used for reporter measurements, reducing the multiparameter dimension of the assay. One solution to this is to increase the number of total parameters that can be measured, thus accommodating additional reporter or encoder probes. For example, mass cytometry [4], a nonoptical flow cytometry approach, uses polymeric lanthanide chelates as probes enabling 40 or more labels to be measured simultaneously on individual particles, and these can be used either for reporting or encoding [5]. For more conventional optical detection, spectral flow cytometry [6-8] can enable more efficient use of the optical spectrum and improve the measurement of fluorophores that have a high degree of spectral overlap, increasing the number of fluorophores that can be used simultaneously. This could be especially useful for implementing the fluorescent protein-based barcoding demonstrated by Smurthwaite et al. (this issue, page 105), as the popular fluorescent proteins used in this approach have significant spectral overlap with popular antibody labels, complicating experimental design on a conventional flow cytometer. Spectral flow cytometry also enables the use of new types of probes, such as surface enhanced Raman scattering tags [9] that offer the potential for very high levels of encoding.

Single cell analysis is at the heart of modern biomedical research and drug development, and flow cytometry is a key analytical platform, offering the potential for highly multiparameter and multiplexed measurements of complex cell systems. Encoding approaches such as the FP barcoding presented by Smuthwaite are gaining increased popularity and, when merged with the highly multiparameter reporter measurements for which flow cytometry is known, and the high throughput measurement capabilities recently developed for flow cytometry [10], make extremely powerful tools for studying complex cell systems.

LITERATURE CITED

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