Fluorescent cell barcoding in flow cytometry allows
high-throughput drug screening and signaling profiling
Peter O Krutzik & Garry P Nolan
Flow cytometry allows high-content, multiparameter analysis of
single cells, making it a promising tool for drug discovery and
profiling of intracellular signaling. To add high-throughput
capacity to flow cytometry, we developed a cell-based
multiplexing technique called fluorescent cell barcoding (FCB).
In FCB, each sample is labeled with a different signature, or
barcode, of fluorescence intensity and emission wavelengths, and
mixed with other samples before antibody staining and analysis
by flow cytometry. Using three FCB fluorophores, we were able to
barcode and combine entire 96-well plates, reducing antibody
consumption 100-fold and acquisition time to 5–15 min per
plate. Using FCB and phospho-specific flow cytometry, we
screened a small-molecule library for inhibitors of T cell–
receptor and cytokine signaling, simultaneously determining
compound efficacy and selectivity. We also analyzed IFN-c
signaling in multiple cell types from primary mouse splenocytes,
revealing differences in sensitivity and kinetics between B cells,
CD4+ and CD4– T cells and CD11b-hi cells.
Flow cytometry is a high-content, multiparameter platform that
allows analysis of multiple antigens at the single-cell level. Current
instrumentation permits the simultaneous analysis of ten or more
parameters, including light-scatter characteristics and fluorescence
measurements. Flow cytometry assays are available to measure
concentrations of intracellular cytokines and proteins, DNA con-
tent, calcium flux, apoptosis, and amount of membrane-bound
extracellular surface markers and many other cell-associated anti-
gens as well as other cellular processes1,2.
With phospho-specific flow cytometry, or phospho flow, it is
possible to measure the phosphorylation status of proteins critical
to intracellular signaling cascades at the single-cell level3–5. Owing
to the multiparameter nature of flow cytometry, many phospho-
of multiple upstream and downstream signalingcomponents with-
inindividualcells6.Thus,onecan effectively monitor theselectivity
of stimuli and drugs for particular intracellular signaling pathways.
Because surface markers can be measured simultaneously with
phospho-proteins, phospho flow is ideally suited to analysis of
complex, heterogeneous populations of cells, such as peripheral
blood lymphocytes or mouse splenocytes7–11, both in vitro and
in vivo12,13, as well as for large-scale profiling of intracellular
signaling biosignatures in both normal and disease states13,14.
The technique also has great potential for drug screening and in
late-phase clinical monitoring of drug efficacy in patients3,15,16.
But as the scale of flow cytometry experiments expands from a
few dozen samples to hundreds or thousands of samples in a high-
throughput screen, several limitations are encountered, including
antibody reagent expense and sample acquisition throughput.
Throughput has been increased by rapid auto-samplers, but these
are not widely available17,18. In addition, phospho flow, like any
plate-based assay, can suffer from staining variability among
samples as a result of inconsistency in sample volume or addition
of staining reagents.
One possible answer to these high-throughput screening pro-
blems is to multiplex samples, that is, to combine multiple samples
into one analysis tube before staining. To date, several techniques
have been developed for multiplexing on the flow cytometry
platform, but nearly all are based on bead technology19–22.
Here we present a cell-based multiplexing approach, FCB, which
dramaticallyreduces antibody consumption, improves the
throughput of flowcytometryexperiments,andeliminates staining
variability between samples. In the FCB technique, samples are
labeled with different intensities of a fluorophore (FCB marker) by
treatment with different concentrations of the reactive form of the
fluorophore. This gives each sample a unique signature of fluores-
that, the number of different fluorescent signatures available
increases geometrically because of multiplexing of fluorescence
intensity with fluorescence emission wavelength (for example,
using different channels on the flow cytometer). Using one fluor-
ophore as an FCB marker, 4–6 different samples can be barcoded
and analyzed together. By using FCB reagents in a combinatorial
fashion, 16–36 samples can be barcoded with two FCB fluoro-
phores, 64–216 with three, and so on.
We demonstrate application of the technique to barcoding of 96
samples simultaneously with excellent resolution, both in cell lines
and in primary mouse splenocytes. Antibody consumption was
96-well plate. These advances make large-scale drug screening and
disease signaling profiling more feasible on the flow cytometry
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RECEIVED 4 JANUARY; ACCEPTED 10 MARCH; PUBLISHED ONLINE 20 APRIL 2006; DOI:10.1038/NMETH872
Department of Microbiology and Immunology, Baxter Laboratory in Genetic Pharmacology, Stanford University, Stanford, California 94305, USA. Correspondence
should be addressed to G.P.N. (email@example.com).
NATURE METHODS | VOL.3 NO.5 | MAY 2006 | 361
bandpass filter for Cascade Yellow with a 610/20 bandpass filter for
detection of Quantum Dot 605.
Additional methods. Description of determination of FCB marker
concentration and calculations of fold change, percent inhibition
and Z scores are available in Supplementary Methods.
Note: Supplementary information is available on the Nature Methods website.
We thank E. Danna and R. Wolkowicz for critical reading of the manuscript and
J. Crane for technical support. P.O.K. was supported by a Howard Hughes Medical
Institute predoctoral fellowship. G.P.N. was supported by National Heart Lung
and Blood Institute contract N01-HV-28183 and National Institutes of Health
COMPETING INTERESTS STATEMENT
The authors declare competing financial interests (see the Nature Methods website
Published online at http://www.nature.com/naturemethods/
Reprints and permissions information is available online at
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