Fluorescent Cell Barcoding (FCB) was designed as a way to enable higher throughput flow cytometry while minimizing reagent consumption and maximizing data robustness (Krutzik and Nolan, 2006
). The FCB technique encodes different cell samples with unique fluorescent signatures and combines the samples together for simultaneous antibody staining and data acquisition. This eliminates sample-to-sample variation arising from differences in staining volume and antibody concentration. In addition, because many samples are combined into one tube/well for acquisition, acquisition times are reduced, enabling entire 96 well plates to be run in 5–10 minutes without autosamplers. These advantages enable researchers to perform larger screening experiments, both for drug discovery and basic research in immunology, than would previously have been possible with standard one tube, one stain methods.
In FCB, cells are labeled with unique signatures or “barcodes” of fluorescent dyes. The fluorescent dyes are derivatized with N-hydroxysuccinimide and are therefore reactive to amine functional groups present primarily on protein lysine side chains and at the N-terminus. By staining cell samples with different concentrations of reactive fluorescent dye it is possible to impart samples with unique dye intensity distributions. Since reacted dye is covalently attached to the cells, non-reacted dye can be washed away enabling mixing of differentially labeled samples into one tube for antibody staining. The samples are distinguishable during software analysis based on their fluorescence intensity in the barcoding channel. Many different dyes are amenable to FCB, which permits multiparameter barcoding. The use of multiple FCB parameters facilitates high level multiplexing. For example, two parameters at four levels of barcoding each encode 16 populations (4 × 4), three parameters at four levels each encode 64 populations (4 × 4 × 4), and so on. We have previously used three dyes to encode an entire 96 well plate (in a 6 × 4 × 4 matrix) (Krutzik and Nolan, 2006
The FCB method is typically used in experiments where cells have been fixed and permeabilized for analysis of intracellular cytokines or phospho-proteins (Krutzik and Nolan, 2003
). This is preferable because the amine-reactive dyes used find orders of magnitude more protein targets inside the cell as compared to outside the cell. This enables higher level of barcoding intensity with lower amounts of dye when cells have been permeabilized. However, FCB of live cells is possible (manuscript in preparation).
Because of its ability to reduce antibody consumption, increase throughput, and minimize staining variability, the FCB method is well suited for experiments involving profiling of many samples, e.g. drug discovery, disease profiling, patient monitoring, and method optimization. The FCB platform is useful for both cell lines and primary cell samples such as human peripheral blood or murine splenocytes (Krutzik and Nolan, 2006
). Among its applications, the method has enabled high-content drug screening in primary cells (Krutzik et al., 2008
), profiling of lymphoma cell signaling (Irish et al., 2010
), and more effective signal amplification of phospho-protein signaling using enzyme-mediated reporter deposition (Clutter et al., 2010
This unit contains two Basic Protocols. The first applies FCB to a cell line, using one dye to barcode four samples. The second protocol uses three dyes to barcode 27 primary peripheral blood samples. Although particular dyes are used in these protocols, the methods are readily adaptable to use of other fluorescent dyes. Support Protocols for each of the Basic Protocols outline effective software analysis of the barcoded data, a critical step to application of the method.