Optical coding of mammalian cells using semiconductor quantum dots

Cell-based assays are widely used to screen compounds and study complex phenotypes. Few methods exist, however, for multiplex- ing cellular assays or labeling individual cells in a mixed cell population. We developed a generic encoding method for cells that is based on peptide-mediated delivery of quantum dots (QDs) into live cells. The QDs are nontoxic and photostable and can be imaged using conventional Xuorescence microscopy or Xow cytometry systems. We created unique Xuorescent codes for a variety of mammalian cell types and show that our encoding method has the potential to create 1100 codes. We demonstrate that QD cell codes are compatible with most types of compound screening assays including immunostaining, competition binding, reporter gene, receptor internalization, and intracellular calcium release. A multiplexed calcium assay for G-protein-coupled receptors using QDs is demonstrated. The ability to spectrally encode individual cells with unique Xuorescent barcodes should open new opportunities in multiplexed assay development and greatly facilitate the study of cell/cell interactions and other complex phenotypes in mixed cell populations.

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 327 (2004) 200–208 www.elsevier.com/locate/yabio Optical coding of mammalian cells using semiconductor quantum dots Larry C Mattheakis,Ô Jennifer M Dias, Yun-Jung Choi, Jing Gong, Marcel P Bruchez, Jianquan Liu, and Eugene Wang Quantum Dot Corp., 26118 Research Road, Hayward, CA 94545, USA Received 25 September 2003 Abstract Cell-based assays are widely used to screen compounds and study complex phenotypes Few methods exist, however, for multiplexing cellular assays or labeling individual cells in a mixed cell population We developed a generic encoding method for cells that is based on peptide-mediated delivery of quantum dots (QDs) into live cells The QDs are nontoxic and photostable and can be imaged using conventional Xuorescence microscopy or Xow cytometry systems We created unique Xuorescent codes for a variety of mammalian cell types and show that our encoding method has the potential to create 1100 codes We demonstrate that QD cell codes are compatible with most types of compound screening assays including immunostaining, competition binding, reporter gene, receptor internalization, and intracellular calcium release A multiplexed calcium assay for G-protein-coupled receptors using QDs is demonstrated The ability to spectrally encode individual cells with unique Xuorescent barcodes should open new opportunities in multiplexed assay development and greatly facilitate the study of cell/cell interactions and other complex phenotypes in mixed cell populations  2004 Elsevier Inc All rights reserved Keywords: Quantum dots; Multiplexed cell-based assays; Spectral encoding; Receptor; Nanocrystal The growing size of compound libraries and therapeutic targets has driven the need for new screening technologies The desire to develop new methods for massively parallel analyses has led to the development of microarray chips [1–5] and encoded microsphere beads [6–11] for use as biosensors and for studying nucleic acids and proteins These methods have been useful for studying biochemical interactions, but there has been limited progress to extend these approaches to cell-based screening Cell-based assays are widely used to screen the activities of compounds against important membrane receptor targets or to provide important preclinical data on a compound’s toxicity or bioavailability To multiplex cell-based assays, it is possible to use positional cell arrays, but these systems require sophisticated robotic systems or unique substrate surfaces that are cell-type speciWc Cell patterning via surface modiW- cation of the substrate can be accomplished by chemical, photochemical, or lithographic methods [12–16] Microfabrication of nanowells on a membrane surface has also been used to construct cell microarrays [15] An alternative approach, transfected cell microarray, is based on culturing mammalian cells on glass slides printed with deWned cDNAs [17] The cells take up the DNA and create deWned locations of transfected cells on the slide surface To create a more versatile multiplexing strategy for cell-based assays, it would be desirable to encode individual cells with unique identiWer barcodes Such a system could then be used for a variety of cell types and would not require that cells adhere to an array surface Encoded cells would also be compatible with standard single cell analysis platforms such as microscopy or a Xuorescence-activated cell sorter (FACS).1 Ô Corresponding author Present address: Cytokinetics, Inc., 280 East Grand Ave., South San Francisco, CA 94080, USA; Fax: 1-650624-3010 E-mail address: lmattheakis@cytokinetics.com (L.C Mattheakis) Abbreviations used: FACS, Xuorescence-activated cell sorter; QD, quantum dot; CHO, Chinese hamster ovary; GPCR, G-proteincoupled receptor, HA, hemagglutinin; PBS, phosphate-buVered saline 0003-2697/$ - see front matter  2004 Elsevier Inc All rights reserved doi:10.1016/j.ab.2004.01.031 L.C Mattheakis et al / Analytical Biochemistry 327 (2004) 200–208 Here we describe a cell encoding technology based on quantum dot (QD) nanocrystals QDs are nanometersized crystals of semiconductor material, typically 2– nm in diameter Although they are chemically identical to bulk semiconductor material, they exhibit optical properties that are highly dependent on their size [18] QDs can be excited to emit light in a manner analogous to that of organic Xuorophores Unlike organic dyes, however, their broad absorption spectrum allows all colors to be excited with a single wavelength of light and they not bleach signiWcantly [11,19,20] Recent advances in QD chemistry have made it possible to transfer quantum dots into aqueous buVers and to modify the surface so that biological aYnity molecules such as antibodies and nucleic acid probes can be attached and used as direct labels to detect biological markers in various applications [20–27] QDs have also been used to label live cells The earliest example showed that a transferrin–QD conjugate is transported into live HeLa cells by receptor-mediated endocytosis [23] More recent work has shown that QDs encapsulated in phospholipid micelles can be injected into Xenopus embryos and used to trace cell lineage [26] Finally, multicolor QDs were shown to be useful for visualizing the eVects of diVerent starvation times on aggregation of Dictyostelium discoideum [28] Here we describe a QD system for encoding, imaging, and decoding single cells for multiplexing and other assay applications (Fig 1) We developed a generic method to deliver multicolor QDs into live mammalian cells and show that QDs are compatible with a variety of important drug-screening cell-based assays Each cell type is encoded separately with a unique and spectrally resolvable QD code The encoded cells are mixed and 201 aliquots of the mixture are deposited into the wells of an assay plate We used a microscope-based imaging system to identify and decode individual cells and show examples of using this encoding scheme to multiplex cellbased assays Materials and methods Preparation of water-soluble quantum dots Organic-soluble, CdSe/ZnS core-shell nanocrystals [19,29] were isolated from hexanes and ligand solution with an equal volume of methanol, rinsed with methanol, and redispersed in CHCl3 These materials were mixed with neutralized amphiphilic polymer (40% octylamine-modiWed polyacrylic acid, 2000 units/QD) in CHCl3, and the solvent was evaporated The dry Wlm was redispersed in water and puriWed from excess polymer by gel Wltration The surface coating was crosslinked further with 1-ethyl-3-(3-dimethylamino propyl) carbodimide-mediated coupling to lysine (or polyethylene glycol-lysine) These materials were then puriWed by gel Wltration and ion exchange spun columns in the presence of 10 mM borate buVer, pH 8.2 Delivery of QDs into cells using Pep-1 CHO-KI cells (ATCC) were incubated at 37 °C, 5% CO2 in growth medium consisting of Dulbecco’s modiWed Eagle’s medium/nutrient mixture F12 (DMEM/F12) containing 5% serum Cells were seeded in 35-mm-diameter wells and grown to a density of £ 105 cells per well The Pep-1 peptide is available commercially as Chariot Fig Schematic illustration of a multiplex cellular screening assay using QDs DiVerent cell lines or a common host cell line expressing diVerent receptors are encoded separately with QD cell codes The cells are mixed and aliquots from the master mix are deposited into the wells of a clear-bottomed assay plate Compounds are added to the wells of the assay plate and the plate is imaged using an inverted-microscope-based imaging system Alternatively, the cells are removed from the assay plate and analyzed by FACS 202 L.C Mattheakis et al / Analytical Biochemistry 327 (2004) 200–208 from Active Motif (Carlsbad, CA) To form a complex between QDs and Pep-1, QDs were diluted in phosphate-buVered saline (PBS) (pH 7.2) to a Wnal volume of 0.1 ml and added to an equivalent volume of Pep-1 diluted in water The complex was incubated for 30 at room temperature The cell culture medium was removed, and the 0.2-ml complex was added Wrst to cells, followed by 0.4 ml of DMEM/F12 The cells were incubated for h and ml of DMEM/F12 containing 5% serum was added The cells were incubated for an additional h and either lifted for analysis or incubated overnight as described Cloning and expression of epitope-tagged GPCRs The cDNAs for the
2-adrenergic, serotonin 2A, and serotonin 2B receptors were cloned from human brain mRNA or cDNA libraries (BD Biosciences Clontech, Palo Alto, CA) The sequence for the nine-amino acid hemagglutinin (HA) epitope was inserted at the 50 end of the coding sequence and the genes were cloned into the eukaryotic expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA) CHO cells were transfected and selected in the presence of 800 g/ml G418 Clones expressing receptor were isolated by FACS using a Xuorescent antibody directed against the HA sequence Receptor internalization assay CHO cells expressing the HA-tagged
2 adrenergic receptor were preincubated with a mouse monoclonal anti-HA antibody (CRP Inc., Denver, PA) for 30 Cells were incubated in the presence or absence of agonist (10 M isoproterenol), Wxed with 3.7% formaldehyde in PBS, and permeabilized with 0.1% Triton X-100 in Blotto buVer (3% dry milk, 25 mM Tris–HCl, pH 7.4, 137 mM NaCl, mM KCl, mM CaCl2) The samples were incubated with Xuorescein isothiocyanate-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) for 30 and imaged by Xuorescence microscopy Multiplex calcium assay and imaging Cells were seeded into the wells of a clear-bottomed 96-well plate and loaded with a calcium dye using the FlexStation calcium assay kit (Molecular Devices, Sunnyvale, CA) The cells were imaged using the Discovery-1 microscope system (Universal Imaging Corp., Downingtown, PA) The calcium Xuorescence image of cells was acquired before and approximately s after addition of carbachol (30 M Wnal concentration) To determine the QD codes, the same Weld of view was imaged with 15-nm-bandwidth emission Wlters centered at 560, 575, 590, 605, 620, and 635 nm The calcium Xuorescence fold induction was measured using the Metamorph V version 4.5 software package (Universal Imaging Corp.) Results Transfecting QDs into live cells To deliver quantum dots into live cells, we explored the use of peptide translocation domains, cationic lipids, and polymeric micelles All of these methods resulted in the internalization of quantum dots, but the percentage of labeled cells and eVects on cell viability varied depending on the method and QD surface properties These results demonstrate that a variety of methods exist for labeling cells with QDs We found that the protein translocation domain Pep-1 is an eYcient and convenient carrier for delivering QDs into cells Pep-1 is a synthetic 21-residue amphipathic peptide composed of three functional sequences: a hydrophobic tryptophan-rich sequence, a spacer sequence, and a hydrophilic lysine-rich sequence from the nuclear localization sequence of simian virus 40 large T antigen [30] The Pep-1 peptide is nontoxic and has been shown to deliver a variety of peptides and proteins into cells [30] The hydrophobic sequence binds to proteins or QDs noncovalently, and the lysine-rich sequence functions to penetrate cells and deliver the bound complex intracellularly We incubated a Wxed concentration of Pep-1 with increasing concentrations of QDs and tested each complex for its ability to penetrate Chinese hamster ovary cells as measured by a FACS Fig 2A shows that the mean cell Xuorescence reaches a maximum at nM for a 560-nm-emitting QD but then decreases to near background levels at 10 nM A similar bell-shaped curve was observed for other QD materials, but the absolute optimum QD concentration varied depending on the speciWc QD material used in the experiment (data not shown) Fig 2A also indicates that QD penetration into cells is dependent on the carrier Pep-1 To study this concentration dependence in more detail, we Wxed the 560-nm QD concentration at 10 nM and measured the eVect of increasing peptide concentration Fig 2B shows that doubling the QD concentration required a concomitant twofold increase in peptide concentration to obtain the maximum increase in cellular Xuorescence Increasing the peptide concentration beyond 60 M did not increase QD internalization Together, these results suggest that multiple copies of carrier peptide are associated per QD and that an optimum number is required for eYcient delivery into cells This conclusion is supported by a previous study that showed, for green Xuorescent protein and
-galactosidase, that a molar ratio of Pep-1:protein of 40:1 was optimal for delivery into Cos-7 or HS-68 cells [30] L.C Mattheakis et al / Analytical Biochemistry 327 (2004) 200–208 203 Fig EVect of varying QD (560 nm) or Pep-1 concentration on QD delivery into cells (A) QDs were incubated in the presence (squares) or absence (triangles) of 30 M Pep-1 (B) Pep-1 was incubated in the presence (squares) or absence (triangles) of 10 nM QDs The complexes were added to CHO cells as described under Materials and methods The cells were incubated overnight and lifted, and the Xuorescence intensity was measured by FACS The mean Xuorescence units are the average of 10,000 events Our data estimates an optimized Pep1:QD ratio, for CHO cells, of approximately 6000:1 for the 560-nm QD material shown in Fig We also measured the percentage of QD-labeled cells by Xuorescence microscopy Nearly 100% of CHO cells contain QDs under these optimized conditions The QDs are contained within vesicles, but the number of QDcontaining vesicles and the total Xuorescence intensity varies among individual cells Confocal microscopy conWrmed that the QDs are intracellular and that the vesicles are distinct from the mitochondria and endoplasmic reticulum compartments (data not shown) The Pep-1 transfection method for QDs is not limited to CHO cells We used the same procedure to introduce QDs into a variety of mammalian cell types, but the QD concentration had to be optimized for each cell type We demonstrated QD transfection of HEK293, NIH3T3, SKBR3, and the primary cell line HUVEC and found the percentage of transfected cells to range from 80 to 95% under optimized conditions We also tracked the stability of QDs inside CHO cells by comparing the growth rates of cells transfected with QDs to nontransfected cells over a 6-day period The growth rates were similar, and the percentage of labeled cells decreased in concordance with the number of cell doublings To measure QD toxicity, we used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT reagent) to measure cell survival after QD transfection [31] Although some QD materials were toxic in this assay, we found that a combination of anionic, cationic, and size exclusion puriWcation of QDs after stabilization could reduce toxicity to near undetectable levels at the concentrations used for transfection Development of cell codes To detect QDs inside cells, it is possible to use a FACS or microscope to measure the intensity and wavelengths of the codes We chose microscopy because it is more amenable to multicolor imaging, and a greater variety of cell-based assays are compatible with microscope-based imaging systems compared to Xow-based systems Our imaging system is a standard epiXuorescence microscope equipped with a single 488-nm argon laser and two emission Wlter wheels containing 18 diVerent Wlters The 10-nm band pass Wlters are evenly spaced to cover the visible region from 510 to 680 nm The Wlters are used to determine the Xuorescence emission spectra of individual cells We devised a decoding algorithm based on comparing the pattern of an unknown cell’s spectrum to stored reference spectra The algorithm is based on the Pearson coeYcient, which describes the strength of the association between the observed values and the predicted reference values [32] A coeYcient of 0.95 was used as a cutoV to match an observed cell’s spectrum to that of the reference To test our imaging and decoding system for resolving codes, we incubated the Pep-1 peptide with Wve single-color or Wve dual-color QD combinations and added the complexes to CHO cells The cells were incubated overnight and the spectra of individual cells determined (Fig 3) Although the absolute Xuorescence intensity of individual cells can vary greatly, the curves are quite similar when the area under the curve of each cell’s spectrum is normalized relative to that of the brightest cell The similarity of the normalized curves for the dual color codes indicates that cells take up diVerent QD colors at nearly the same ratio for a variety of QD combinations We also found that it was possible to vary the concentration ratios of QD colors and obtain additional codes For example, the combination of QD colors 582 and 630 nm at 2:1 or 1:1 molar ratios yielded two diVerent codes as determined by our decoding algorithm (Fig 3) To test multiplexing, we set up a mock Wve-plex assay Five separate cultures of CHO cells were encoded with 566-, 582-, 608-, 630-, or 647-nm QDs The cells were lifted after the encoding step and mixed, and aliquots were deposited into the wells of a 96-well assay plate for imaging the next day (Fig 4A) Fig 4B shows that the mixed population of cells could be segregated into Wve distinct QD codes These results demonstrate that we 204 L.C Mattheakis et al / Analytical Biochemistry 327 (2004) 200–208 Fig Fluorescence emission spectra of 10 QD cell codes CHO cells were encoded as described under Materials and methods and Wxed, and the normalized Xuorescence spectra (510–680 nm) were determined from complexes consisting of 40 M Pep-1 and the indicated QD colors (5 nM each) For the 582- and 630-nm codes, the 2:1 and 1:1 molar ratios for the 582- and 630-nm QDs were nM:2.5 nM and 2.5 nM:2.5 nM, respectively Shown for each code are the normalized spectra of approximately 25 cells Each line is the normalized spectrum of a single cell Fig Imaging and decoding of a mixed cell population (A) CHO cells were encoded with 566-, 582-, 608-, 630-, or 647-nm QDs, mixed, and transferred to an assay plate for imaging the next day Cell nuclei were stained with Hoechst 33342 Shown is an image captured using a Nikon D1 color digital camera Filter sets for excitation and emission were 390 § 100 nm and 490 nm long pass, respectively (B) Normalized emission spectra of approximately 60 cells from the mixed cell population Each line is the normalized spectrum of a single cell can encode, image, and decode mixed cell populations in the absence of an assay Encoded cellular assays To use QDs for encoding cellular assays, the internalized QDs must not interfere with normal cell physiology such as signal transduction, receptor traYcking, and membrane function We tested the eVects of QDs in a variety of cell-based assays including immunostaining, receptor binding, reporter gene expression, receptor internalization, and intracellular release of calcium To test the eVect of QDs in an immunostaining application, we encoded CHO cells with 530-nm QDs The cells were Wxed and stained using a Xuorescent antibody directed against tubulin (Fig 5A) Control experiments showed that the staining intensity was equivalent in the presence or absence of QDs (data not shown) We also tested the eVect of QDs on binding of a Xuorescent ligand to a cell surface receptor CHO cells expressing the
2-adrenergic receptor were encoded with 530-nm QDs and prepared for a competition-binding assay The Xuorescent ligand CGP-12177 bound to cells expressing the receptor (Fig 5B), although the intensity of CGP-12177 Xuorescence among individual cells varied, possibly due to diVerences in receptor expression or quenching of CGP-12177 Xuorescence by the QDs Binding was blocked in the presence of excess unlabeled CGP-12177 (Fig 5C) Therefore, these results indicate that internalized QD codes not interfere with binding of ligands or antibodies to intracellular and membrane surface targets The presence of intracellular vesicles containing QDs could potentially aVect protein traYcking To test this, we measured the agonist-induced internalization of the
2 adrenergic receptor CHO cells encoded with 608-nm QDs and expressing an epitope-tagged version of the
2 receptor were incubated in the absence (Fig 5D) or presence (Fig 5E) of the agonist isoproterenol Fig 5E shows that isoproterenol causes the
2 receptor to trans- L.C Mattheakis et al / Analytical Biochemistry 327 (2004) 200–208 205 Fig Fluorescent microscopy images of encoded cells in various cellular assays (A) Immunostaining of tubulin in CHO cells Cells were encoded with 530-nm QDs and Wxed for immunostaining using rabbit antitubulin IgG fraction (Sigma–Aldrich, St Louis, MO), biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA), and streptavidin-conjugated Cy3 (Amersham Bioscience, Piscataway, NJ) Shown is a composite of the Cy3 and QD images (B and C) Binding of CGP-12177 to CHO cells expressing the
2-adrenergic receptor CHO cells were encoded with 530-nm QDs and incubated with 250 nM BODIPY TMR (§) CGP-12177 (Molecular Probes, Eugene, OR) in the absence (B) or presence (C) of M unlabeled CGP-12177 (Sigma–Aldrich) Shown are composites of the QD and BODIPY images Binding was measured as total pixel intensity of CGP-12177 Xuorescence (D and E) Agonist-induced internalization of the
2-adrenergic receptor CHO cells expressing HA-tagged
2-adrenergic receptor were encoded with 608-nm QDs and incubated in the absence (D) or presence (E) of 10 M isoproterenol The cells were assayed for receptor internalization as described under Materials and methods Shown is a composite of the FITC and QD images locate from the cell surface to intracellular vesicles of the encoded cells To determine the dose response of translocation, we measured the intensity of intracellular Xuorescence and found the EC50 values to be similar in encoded and unencoded cells (data not shown) Thus, QDs contained within vesicles appear to be relatively inert and not interfere with traYcking events within the cell To test the eVects of QDs on signal transduction, we used a CHO cell line expressing the
2-adrenergic receptor and carrying a luciferase reporter gene under the transcriptional control of a cyclic adenosine monophosphate (cAMP) responsive promoter Stimulation of the
2 receptor with the agonist isoproterenol results in elevation of cAMP, which activates transcription of the luciferase reporter We found the dose responses in unencoded cells and encoded cells to be similar and the EC50 values were approximately nM (Fig 6) Finally, to demonstrate the use of QDs in a multiplexing experiment, we encoded three diVerent GPCR cell lines and simultaneously measured the agonist-induced eVects on intracellular calcium levels Cell lines expressing the muscarinic M1, serotonin 2A, and serotonin 2B receptors were encoded with 605-, 582-, and 630-nm QDs, respectively The cell lines were mixed, and aliquots of the mixture were transferred into the wells of an assay 206 L.C Mattheakis et al / Analytical Biochemistry 327 (2004) 200–208 Fig EVect of QDs on a luciferase dose response assay Approximately £ 105 of control or 530-nm QD-encoded CHO cells expressing the
2-adrenergic receptor and renilla luciferase reporter gene were seeded into each well of a 96-well assay plate Cells were incubated overnight in DMEM/F12 medium containing 10% serum The cells were washed and incubated 24 h in DMEM/F12 medium lacking serum or phenol red Cells were incubated with isoproterenol at the indicated concentrations for h, washed, and assayed for luciferase activity using the renilla luciferase assay system (Promega Corp., Madison, WI) and SpectraFluor Plus instrument (Tecan, Research Triangle Park, NC) Shown are the dose responses of encoded (dashed line) and control (solid line) cells Values are the average of three measurements plate The next day, the cells were loaded with a Xuorescent calcium indicator and incubated in the absence (Fig 7A) or presence (Fig 7B) of carbachol, a speciWc agonist of the muscarinic receptor Figs 7A and B show that carbachol increased calcium Xuorescence of speciWc cells within the population We randomly chose 30 cells that responded to carbachol and determined the average increase in calcium Xuorescence intensity to be 3.2-fold (Fig 7C) The 30 responsive cells were imaged with a series of emission Wlters, and all were found to contain the 605-nm QD codes We also imaged 30 cells each expressing the serotonin 2A or 2B receptors and found the average fold-increase in calcium Xuorescence to be 1.0 and 1.1-fold, respectively (Fig 7C) Control experiments showed that the agonist serotonin had no eVect on the muscarinic receptor Thus, these results indicate that the calcium responses of individual cells can be assayed within a mixed cell population and that QDs can be used to encode and multiplex a functional cell-based assay Discussion We have developed a generic method of encoding cells for use in a variety of assays The QD codes can be used with diVerent cell types, and they not appear to aVect the cell’s physiology under the assay conditions that we tested QD cell codes can be used to potentially multiplex virtually any microscope- or FACS-based cellular assay with an optical readout The readout can be Xuorescence, luminescence, or bright-Weld imaging For a Xuorescent reporter, such as an organic dye that senses intracellular calcium, the codes can be chosen to minimize spectral overlap with the reporter Although we demonstrated encoding of calcium, reporter gene, receptor internalization, and competition binding assays, QD cell codes could also be used to multiplex other existing assays such as toxicity, apoptosis, neurite outgrowth, and membrane potential This encoding platform may also enable new assays where it is essential that multiple cell types be barcoded and observed within a complex and mixed population For example, certain types of motile tumor cells have been shown to engulf QDs [33], and it may be possible to use this process to introduce unique QD codes into multiple tumor cell types and observe their motility simultaneously within a mixed population Another application is based on recent results showing that QDs and DNA can be cotransfected into cells using cationic lipids (L.C Mattheakis, unpublished data) Thus, it may be possible to encode DNA transfections and observe the behavior of the transfected cells in suspension rather than on an array surface Many of these applications are not easily achieved using organic dyes or other encoding formats Organic dyes provide a limited number of color choices, and their spectral overlap further decreases the number of useful codes The photostability of organic dyes varies widely, and this may complicate their use as cell codes when used in diVerent combinations [34] Metallic barcodes, metal nanoparticles that are self-encoded with submicrometer stripes, are being developed for a variety of multiplexing applications, but these particles are submicrometer in size and much larger than mammalian cells [35] QDs can potentially create a large number of cell codes A simple binary encoding strategy (2N ± 1, N D number of resolvable colors) estimates 31 distinct codes using Wve colors Our results, however, suggest that a higher encoding capacity is possible because the combination of QD colors 582 and 630 nm yielded distinct codes when the QD colors were used at diVerent intensity ratios Therefore, using Wve colors, it may be possible to generate codes based on ternary (3N ± 1, 243 codes) or quaternary (4N ± 1, 1023 codes) encoding schemes depending on the optical resolution of the intensity levels Although these numbers may seem small compared to the number of features present on DNA or protein microarrays, they may be more than adequate for most cellular assays Encoded cells also oVer important advantages compared to cell arrays Cell arrays require adherant cells, and the cells must be grown in parallel and in a miniaturized format without cross-contamination The cells must also have access to the necessary nutrients, L.C Mattheakis et al / Analytical Biochemistry 327 (2004) 200–208 207 Fig Multiplex calcium assay using QDs CHO cells expressing the muscarinic M1 receptor (ATCC, Manassas, VA) and serotonin 2A and serotonin 2B receptors (see Materials and methods) were encoded separately with 608-, 582-, and 630-nm QDs, respectively The cells were mixed, and aliquots were transferred to the wells of a 96-well clear-bottomed assay plate The calcium assay and imaging were as described under Materials and methods Shown are the same Weld of view images of cells incubated in the absence (A) or presence (B) of 30 M carbachol Some cells are circled and identiWed as examples The images are composites of the calcium dye and QD Xuorescent images (C) EVect of carbachol on the cellular calcium response For each receptor type, the calcium dye Xuorescence intensities of 30 cells within the mixed population was measured before and after addition of carbachol to determine the average fold response The error bars indicate standard deviation values chemical compounds, and macromolecules required for cell growth under a variety of conditions Linking the codes directly to single cells allows the cells to be grown and analyzed using well-established and routine cell analysis methods In conclusion, we have created a generic method for encoding single cells The applications for this technology include multiplexing cell-based assays for drug screening and studying mixed cell populations The ability to tag individual cells with unique Xuorescent barcodes should greatly facilitate the study of cell/cell interactions and other complex phenotypes Acknowledgments We thank Stephen Rees (GlaxoSmithKline) for kindly providing the CHO cell line expressing the
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