A universal nanoparticle cell secretion capture assay

A Universal Nanoparticle Cell Secretion Capture Assay

Wendy Fitzgerald,1 Jean-Charles Grivel1,2*

� AbstractSecreted proteins play an important role in intercellular interactions, especially betweencells of the immune system. Currently, there is no universal assay that allows a simplenoninvasive identification and isolation of cells based on their secretion of various pro-ducts. We have developed such a method. Our method is based on the targeting, to thecell surface, of heterofunctional nanoparticles coupled to a cell surface-specific antibodyand to a secreted protein-specific antibody, which captures the secreted protein onthe surface of the producing cell. Importantly, this method does not compromisecell viability and is compatible with further culture and expansion of the secreting cells.Published 2012 Wiley-Periodicals, Inc.y

� Key termscytokine; secretion; capture; nanoparticle

INTRODUCTIONCurrently, immunohistochemistry and flow cytometry are the two main techni-

ques allowing the identification of individual cells secreting a particular protein.

However, because both techniques identify secretory proteins inside the cell, they do

not distinguish between cells that actually secrete these proteins from cells that only

store them (1–3). Moreover, identification of cells which harbor potentially secreted

proteins by flow cytometry requires additional manipulations that include artificial

blocking of the secretory pathway to accumulate the secreted protein inside the cell

and the permeabilization of the cell membrane (4,5), and thus compromises cell via-

bility. However, several secretion-capture assays have been reported earlier. These

assays rely on encapsulating living individual cells in a matrix, which captures and

concentrates secreted proteins, allowing the detection of these proteins on the surface

of the secreting cell. These affinity matrixes were made of gels immobilizing antibo-

dies against a secreted protein of interest (6–8). Another approach to identify the

secreting cells was developed by Brosterhus et al. (9). It consists of generating bispeci-

fic monoclonal antibodies that bind simultaneously to a cell surface antigen and to

the secreted protein of interest by coupling large amounts of pure antibodies of dif-

ferent specificities (9). For the vast majority of investigators, these methods are too

cumbersome.

Here, we report on a novel, easy, inexpensive, and versatile method, which

allows the identification of living cells actually secreting any protein of interest. Our

method is based on the targeting, to the cell surface, of nanoparticles, which capture

the secreted protein on the surface of the secreting cell. This method allows further

characterization of a secreting cell by multicolor flow cytometry and does not com-

promise cell viability.

MATERIAL AND METHODS

Coupling of Magnetic Nanoparticles

A total of 1 mg of carboxyl terminated magnetic iron oxide nanoparticles

(MNPs) (Ocean NanoTech, Springdale, AR) of various diameters (15–25 nm) were

1Section on Intercellular Interactions,Program in Physical Biology, The EuniceKennedy-Shriver National Institute ofHealth and Human Development,Bethesda, Maryland 208922Center for Human Immunology, NationalInstitutes of Health, Bethesda, Maryland20892

Received 2 May 2012; Revision Received17 July 2012; Accepted 15 August 2012

Grant sponsors: The Eunice Kennedy-Shriver National Institute of Health andHuman Development, and the Center forHuman Immunology, National Institutesof Health, Bethesda, MD.

*Correspondence to: Jean-CharlesGrivel, Section on IntercellularInteractions, Program in PhysicalBiology, The Eunice Kennedy-ShriverNational Institute of Health and HumanDevelopment, Bethesda, MD 20892, USA

Email: [email protected]

Published online in Wiley Online Library(wileyonlinelibrary.com)

DOI: 10.1002/cyto.a.22199

Published 2012 Wiley-Periodicals, Inc.y

This article is a US government workand, as such, is in the public domain inthe United States of America.

Brief Report

Cytometry Part A � 00A: 000�000, 2012

coupled to 2 mg (50 ll of a 40 mg/ml solution) of goat anti-

mouse (GAM) purified antibodies (SouthernBiotech, Bir-

mingham, AL), using the manufacturer’s coupling reagents

and buffers kit and following the kit’s protocol. Briefly, the

MNPs are activated using carbodiimide and N-hydroxysucci-

nimide followed by conjugation to amino groups that are

present on the target protein. One mg of MNPs was activated

in 400 ll of activation buffer supplemented with 1.7 mM 1-

(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

(EDC) and 0.76 mM N-hydroxysulfosuccinimide (Sulfo-NHS)

for 20 min at room temperature. After activation, 500 ll ofcoupling buffer was added to the particles, immediately fol-

lowed by the addition 2 mg of the purified antibody. The cou-

pling was allowed to proceed for 2 h in a thermomixer at

room temperature with gentle mixing. The reaction was

stopped by adding 10 ll of quenching solution and transferred

to a 12 3 75 mm2 tube. Two wash-steps with wash/storage

buffer were performed using a SuperMAG-01 magnetic sepa-

rator (Ocean NanoTech) at 48C. The coupled MNPs were sus-

pended in 4 ml of storage buffer and stored at 48C to a final

concentration of 0.25 mg/ml of iron oxide. The superpara-

magnetic property of the MNPs is inversely proportional to

the particle size. Therefore, while 25 nm show superparamag-

netic properties, the cumulative exposure to the high magnetic

fields used in the separation steps of the coupling and the for-

mation of Ab-MNPs complexes preparation (see below) may

result in magnetization of the MNPs, leading to particle aggre-

gation. Therefore, we recommend using 15 nm MNPs.

Coupling of Carboxyl Quantum Dots

A total of 1 nmol of carboxyl terminated quantum dots

with emission 620 nm QD620 (Ocean NanoTech) was conju-

gated to 1 mg (25 ll of a 40 mg/ml solution) of GAM purified

antibodies (Southern Biotech), using the chemicals, buffers,

and Qdots provided with the Qdot conjugation kit following

the kit’s protocol. Briefly, 125 ll QDots was activated by add-

ing 300 ll reaction buffer, after thorough mixing the antibody

solution was added, followed by 50 ll of EDC. The reaction

was allowed to continue for 2 h with gentle mixing at RT in a

thermomixer. The reaction was stopped by addition of 10 llquenching solution. Excess antibody and salt solution were

removed by centrifugation in a 300 KDa molecular weight cut-

off concentrator MWCO (PALL, Port Washington, NY), fol-

lowed by two wash-steps with wash/storage buffer. QDots

were resuspended in 1 ml of wash/storage buffer and stored at

48C

Preparation of Precomplexed MNPs or QDots

Precomplexes were prepared by incubating 100 ll of

GAM coupled magnetic particles, or 50 ll GAM coupled

QDots, 6 lg total of mouse antibodies (typically 3 lg of anti-

cell surface targeting antibody such as anti-CD45 plus 3 lganti-cytokine antibody) for 30 min at RTwith occasional gen-

tle shaking. Antibody-particle complexes were separated from

unbound antibody by one of two methods, magnetic columns

or MWCO concentrators. Magnetic separation is accom-

plished by adding particle/antibody solution to equilibrated

MACS MS columns in a MACS magnet (Miltenyi Biotec,

Auburn, CA).The column is washed three times with MACS

buffer (PBS 2 mM EDTA, 0.2% BSA), and particles are col-

lected by removing the column from the magnet and flushing

with 200 ll MACS buffer (23 the starting volume of parti-

cles). Alternatively particles were separated by centrifugation

in 300 KDa MWCO concentrators at 4,000 rpm in microcen-

trifuge, followed by two wash steps with PBS and then col-

lected in 200 ll of MACS buffer.

Cell Cultures

Whole blood was obtained from the NIH Blood Bank per

their protocol, and peripheral blood monocytes (PBMC) were

isolated using lymphocyte separation medium (Lonza, Walk-

ersville, MD). PBMC were placed in culture overnight in

RPMI 1640 (Invitrogen) supplemented with gentamicin, fun-

gizone (Invitrogen), and 10% fetal bovine serum (Gemini Bio-

products, West Sacramento, CA). PBMC were either nonacti-

vated or activated for 5 h to 2 days with phorbol 12-myristate

13 acetate-PMA (5 ng/ml) and Ionomycin (500 ng/ml)

(Sigma-Aldrich, St Louis, MO). Immediately before use in

experiments, cells were stained with Live/Dead Fixable Blue

dye (Invitrogen, Life Technology, Grand Island, NY) for 15

min at 48C and washed with cold PBS.

Particle to Cell Targeting

To demonstrate the ability of the MNPs to bind specifi-

cally rather than nonspecifically to cells, we prepared precom-

plexes of 15 nm GAM-coupled MNPs with an anti-human

CD3AlexaFluor 488 (eBioscience, San Diego, CA) Ab, mouse

IgG PE (Invitrogen), or both anti-CD3 and msIgG together. A

total of 40 ll MNPs-Ab complex was bound to 13 106 PBMC

in 100 ll PBS with 1% NMS and 1% NGS (Sigma) for 20 min

at 48C, washed with 2 ml cold PBS and spun down at 400g for

5 min to remove any unbound beads, then fixed in 1% para-

formaldehyde-PFA (Electron Microscopy Sciences, Hatfield,

PA) in PBS.

Cytokine Secretion Assays

PBMC were activated with PMA/ionomycin and evalu-

ated for their ability to secrete IL-2 or IFN-c after 5 h, MIP-1aand MIP-1b after 18 h, and RANTES after 2 days. Precom-

plexes were prepared with 15 nm MNPs or Quantum dots and

equivalent amounts of mouse anti-human CD45 eFluor450

(eBioscience) as a targeting antibody to bind to all leukocytes

and mouse anti-human cytokine capture antibody [IL-2, IFN-

c, MIP-1a, MIP-1b, or RANTES (R&D Systems, Minneapolis,

MN)]. A total of 40 ll complexed MNPs were incubated with

1 3 106 cells in 100 ll 1% NMS 1% NGS in PBS for 15 min at

48C with occasional gentle shaking. The cell/bead complexes

were washed with 2 ml cold PBS, spun down, and diluted to

250,000 cells/ml in 4 ml of warm culture medium in a FACS

tube and incubated at 378C for 45 min with constant mixing.

After incubation, cells were spun down, washed at 48C with 2

ml cold PBS, and resuspended in 100 ll of cold 1% NMS 1%

NGS in PBS. Cells were incubated with antibodies against cell

surface markers (CD45, CD3, CD4, CD8) and fluorescently

BRIEF REPORT

2 Universal Nanoparticle Cell Secretion Capture Assay

labeled monoclonal anti-cytokine antibodies for 20 min at

48C. Cells were washed in 2 ml PBS and fixed in 1% PFA in

PBS and analyzed by flow cytometry. Flow cytometry gating

strategy involves gating on lymphocytes by FSC vs SSC, dou-

blet discrimination, and live cell selection followed by the

identification of CD451 cells and then CD8 versus anti-cyto-

kine-labeled cells. CD8 is used since CD3 and CD4 are known

to be downregulated with polyclonal stimulation of lympho-

cytes.

Intracellular Cytokine Staining

PBMC activated and nonactivated were treated with Gol-

giPlug (Invitrogen) for the final 4 h of activation for tradi-

tional intracellular cytokine staining. Briefly, 1 3 106 cells in

100 ll 1% NMS, 1% NGS in PBS were incubated with cell sur-

face antibodies for 20 min at RT. Cells were washed with 2 ml

PBS, spun down, and resuspended in 100 ll Cytofix (Invi-

trogen) and fixed for 20 min at RT. Cells were washed with 2

ml PBS, spun down, and resuspended in 100 ll CytoPerm(Invitrogen) with cytokine detection antibodies and incubated

at 48C for 20 min. Cells were washed with 2 ml PBS, spun

down, and resuspended in 1% PFA in PBS and analyzed by

flow cytometry.

Commercially Available Secretion Assay

IL-2 and IFN-c secretion were also verified by analyzing

both activated and nonactivated PBMC using commercial

secretion kits (Miltenyi Biotec). Briefly, 1 3 106 cells were

incubated with 10 ll capture antibody for 10 min then diluted

in 4 ml of culture medium and incubated at 378C for 45 min

with constant mixing. Cells were spun down and resuspended

in 100 ll 1% NMS 1% NGS in PBS and incubated with cell

surface marker antibodies and 20 ll cytokine detection anti-

body for 20 min at 48C. Cells were washed and fixed in 1%

PFA in PBS and analyzed by flow cytometry.

Flow Cytometric Analysis

Samples were acquired on a BD LSRII (Becton Dickinson,

San Jose, CA) equipped with five LASERs 355, 405, 488, 532,

and 638 nm and with 22 detectors. Data were analyzed with

FlowJo software (TreeStar, Ashland, OR). Flow cytometry gat-

ing strategy involves gating on lymphocytes by FSC vs. SSC,

doublet discrimination, and live cell selection followed by

identification of cells fluorescently labeled by antibodies on

MNPs.

Statistical Analysis

Data normality was verified by the Shapiro-Wilk W test.

When the data did not pass the normality test, the nonpara-

metric procedure of Steel-Dwass for multiple comparison was

used to compare distribution across the three groups studied

(secretion-capture assay, intracellular staining, and commer-

cial secretion assay), when only two groups were compared

(secretion-capture assay and intracellular staining) the Wil-

coxon procedure was used. When the data were found nor-

mally distributed (IL-2 related data), we used an ANOVA with

the Tukey-Kramer HSD (honestly significant difference) cor-

rection. All statistical analyses were performed with JMP 9.0

(SAS Institute, Cary, NC).

RESULTS

We coupled carboxylated MNPs of sizes varying from 15

to 25 nm (10) with polyclonal anti-mouse IgG (H1L) goat

antibodies (GAM). The superparamagnetic nature of MNPs

allows their simple separation, in a magnetic field, from

uncomplexed antibodies, while maintaining their colloidal

properties (Fig. 1a). We reasoned that if an MNP could bind

both a cell-targeting antibody and an antibody specific for a

secreted protein (Fig. 1b), they would attach to the cell surface

and capture the cell-secreted product, which could then be

detected on the surface of the secreting cell. For such a strategy

to function, GAM-MNPs, must be bound to two antibodies of

different specificities, and be attached to the cell surface via

one of these antibodies, which recognizes a cell surface anti-

gen, while the second antibody captures the secreted protein

(Fig. 1c).

We demonstrated the feasibility of this strategy by

attaching to the cell surface, an isotype control antibody

complexed together with a cell surface-specific Ab on GAM-

MNPs. Upon incubation with PBMCs, PE-labeled mouse IgG

isotype control complexed to 15 nm GAM-MNPs together

with an AlexaFluor488-labeled mouse anti-human CD3 mAb

was detected on 92 % of CD3 T cells (Fig. 2a). In contrast,

the same isotype control IgG complexed with MNPs without

anti-human CD3 was found only on 2% of CD31 cells. This

demonstrates that GAM-MNPs can target an irrelevant anti-

body to the cell surface when it is combined with a cell sur-

face-specific Ab.

Next, we verified that this strategy was suitable for the

detection of secreted cytokines on the cell surface. We simu-

lated the secretion of IFN-c by adding this cytokine into the

culture medium bathing PBMCs. A total of 15 nm GAM-

MNPs were complexed with anti-IFN-c and anti-CD45 anti-

bodies. Specifically, we incubated human PBMCs with the

purified MNP complexes, and after washing, added recombi-

nant IFN-c to the cells and washed them 15 min later. IFN-cwas detected with a labeled anti-IFN-c detection antibody

belonging to a different complementation group. We revealed

the presence of IFN-c on the surface of 94.1% of cells. With-

out MNPs, only 2.9% of cells were IFN-c positive (Fig.

2b).The latter may represent either cells that adsorbed exoge-

nous IFN-c or constitutively secrete this cytokine. This

experiment confirmed that GAM-MNPs carrying Ab against

a cell surface protein and an Ab specific for a secreted prod-

uct attach to the cell surface and can capture cell-secreted

products.

Finally, we verified that complexed GAM-MNPs could

indeed capture cell-secreted products. We chose IL-2 and IFN-

c as these two cytokines are produced de novo rather than

being prestored inside the cells and thus the results of our

assay for evaluating the fraction of secreting cells can be com-

pared with standard intracellular staining. We prepared com-

plexes of GAM-MNPs with anti-CD45 Ab and either anti-IL-2

or anti IFN-c Ab. These complexes were incubated with

BRIEF REPORT

Cytometry Part A � 00A: 000�000, 2012 3

human PBMCs, which were activated with a polyclonal activa-

tor to stimulate secretion. Using our assay, we found that 16.9

� 4% and 19.3 � 3.6% of T cells were secreting IL-2 and IFN-

c, respectively (n 5 6) (Fig. 3a). Conventional intracellular

staining revealed IL-2 and IFN-c, respectively, on 16.3 � 1.4%

and on 16.7 � 6.2% of T cells, showing that for revealing cells

that secrete these cytokines the two assays were not statistically

different (n 5 6, P [ 0.8 for both cytokines). We compared

our capture assay to commercial IL-2 and IFN-c capture assaysbased on the use of hetero-bispecific antibodies, which

detected IL-2 on 14.7 � 1.8% and IFN-c on 9.3 � 2.05%, of

cells, which were not statistically different from those detected

with our assay (P 5 0.57 for IL-2 and P 5 0.053 for IFN-c, n5 6) (Fig 3a). These results encouraged us to apply our assay

to identify secreting cells that cannot be identified adequately

by intracellular staining because they constitutively store the

secreted protein. We aimed to identify cells that secrete MIP-1a,MIP-1b, or RANTES. We prepared and incubated with PBMCs

MNPs complexed with anti-CD45 antibodies for cell surface

targeting and either anti-MIP-1a, anti-MIP-1b, or anti-

RANTES antibodies for cytokine capture. The capture assay

detected the secretion of each cytokine. MIP-1a was secreted by

19.95 � 3.5% (n 5 6), MIP-1b was secreted by 10 � 1.8% of

cells (n 5 6), and RANTES was secreted by 10.94 � 6.45% of

Figure 1. Preparation of secretion-capture nanoparticles and secretion assay. (a) Coupling of MNPs: Carboxyl terminated magnetic nano-

particles (MNPs) are activated using carbodiimide and N-hydroxysuccinimide followed by coupling to goat anti-mouse(GAM) purified anti-

bodies. The coupled beads are separated from free antibodies on a magnet. The coupled MNPs now serve as a universal mouse antibody

crosslinker. (b) Preparation of GAM-MNPs Ab complexes: Complexes are prepared by incubating GAM coupled MNPs with a cell-surface-

targeting antibody and a secreted protein-capture antibody, for 30 min at room temperature with occasional gentle shaking. Antibody-

MNP complexes are separated from unbound antibody by washing on MACS magnetic columns in a MACS magnet. Columns are

removed from the magnet and complexes are released by flushing the column. (c) Nanoparticle cell secretion capture assay: GAM-MNPs

complexed with cell-surface-targeting antibodies (Y) and secreted-protein capture antibodies (Y) are attached to the surface of cells via the

targeting antibodies and capture secreted proteins (l) as the cell releases it. The presence of the captured secreted protein is revealed by a

fluorochrome-labeled secreted-protein specific antibody of a different complementation group ( ).

BRIEF REPORT


cells (Fig. 3b). Thus, our assay allows the identification of cells

that secrete both de novo synthesized or stored proteins.

The capture assay described above is not limited to the

use of MNPs. Although MNPs offer an undeniable ease of pu-

rification of complexed particles from free antibodies in a

magnetic field, their use is not compatible with the magnetic

separation of the secreting cells, as every cell is magnetically la-

beled. As an alternative, we have used carboxylated quantum

dots whose size is about 15 nm and we have coupled them to

GAM-Ig (H1L). These nanoparticles can be separated from

uncoupled GAM antibodies and later from uncomplexed Ab

by centrifugation on a Nanosep 300 KDa membrane whose

pore opening is 32 nm. Using this approach, the targeted cells

are also labeled by the Qdot used to form the capture layer on

the cell surface, dispensing the need of using labeled targeting

antibody to visualize the cellular targeting. We have performed

such an assay for detecting IL-2 secretion (Fig. 3c) and have

found that the assay was as good as the MNPs assay.

DISCUSSION

To overcome the limitations of intracellular cytokine

staining, several secretion capture assays have been developed.

These assays rely on encapsulating living individual cells in a

gel matrix, which captures and concentrates the secreted pro-

teins, allowing their detection on the secreting cell (6). The

disadvantages of such gel-based affinity matrixes are multiple.

They require a dedicated instrumentation to encapsulate the

cells in the gel (7,8), and they assume that a single cell is

encapsulated per gel droplet, an assumption that is not always

verifiable. The limitations of these original gel affinity matrixes

were overcome by Brosterhus et al. (9), who used bispecific

monoclonal antibodies to simultaneously bind to a cell surface

antigen and to the secreted protein of interest, resolving the

issue of single cell encapsulation. However, for each protein

assayed, new hetero-bispecific antibodies have to be generated

by coupling large amounts of pure antibodies of different spe-

cificities (9),or by recombinant DNA technologies, or by the

formation and selection of proper hybridoma heterocaryons

(11,12). For the vast majority of investigators, these methods

are too cumbersome and impractical. The paucity of commer-

cially available assays based on hetero-bispecific antibodies is a

testimony to the difficulties involved in preparing such

reagents. Also, the majority of the assays that have been devel-

oped and commercialized by biotechnology companies are

limited to proteins of immunological interest.

Although, similar to our approach for forming an affinity

matrix to capture secreted proteins to the cell surface, none of

the previously developed assays affords the flexibility of our

universal matrix. Indeed, the technique we describe above is

virtually limitless provided that pairs of antibodies (or

ligands) specific for the secreted protein(s) of interest are

available even in small quantity. Moreover, the origin of the

antibodies used has little influence on the assay, because the

nanoparticles can be tailored to capture any type of antibody

by coupling the relevant xenotype, isotype, or allotype specific

polyclonal antibodies. As is it the case for MHC-peptide tetra-

mers (13), our secretion capture assay can be adapted to the

use of streptavidin as a bridging agent between biotinylated

targeting and capture antibodies, provided these antibodies

bear only one biotin to avoid concatenation. The assay, whose

principle is reported here, offers the same degree of freedom

in targeting a specific cellular population as it does in meas-

uring the secretion of any protein. By choosing an antibody

specific for a given cellular marker, one can study the secretion

of proteins by the cell population expressing this cellular

marker. However, unlike the commercial assays, the use of

MNPs to form the affinity matrix, precludes magnetic sorting

of the secreting cells, since every cell, whether it secretes or not

the protein of interest, is magnetically labeled. By adapting the

principle of the assay to the use of non-magnetic labeled

nanoparticle, such as Qdots, secreting cells can be readily

identified by the expression of the targeting marker and the

secretion of the protein studied. In this case, secreting cells can

be magnetically sorted as they are in the commercial secretion

kits, by using magnetic beads specific for the label (fluorescent

or not) of the detection antibody. Using Qdots as an affinity

Figure 2. Nanoparticle targeting to cell surface and cytokine

secretion capture. (a) Cell surface targeting of an isotype control

antibody: 15 nm GAM-MNPs complexed with anti-CD3 AF488 and

a PE-labeled isotype control antibody target the latter to the sur-

face of 92% of T cells (left panel), whereas MNPs complexed only

with the PE-labeled isotype control Ab (without cell-targeting Ab)

are revealed on only 2% of T cells (right panel). (b) Capture of IFN-

c on the cell surface: PBMC treated with 15 nm GAM-MNPs com-

plexed with anti-IFN-c and anti-CD45 antibodies were incubated inPBS containing recombinant IFN-c (left panel) in PBS alone (rightpanel). The binding of IFN-c on the cell surface was revealed witha PE-labeled anti-IFN-c antibody of a different complementationgroup and was detected on 94% of cells incubated with IFN-c andon only 2.9% of cells incubated with PBS.

BRIEF REPORT


matrix, offers the additional advantage of performing ratio-met-

ric measurements of the two channels used to monitor cell bind-

ing and cell secretion, which may allow the normalization and

comparison of protein secretion at the single cell level. However,

such application is out of the scope of this report. The versatility

of the technique we describe here allows the identification of vir-

tually any cells based on the proteins they secrete. The simplicity

of the technique and the ease of its application bring this ability

outside of the traditional realm of well-equipped laboratories

focused on molecules of immunological interest.

Figure 3. Cell secretion assay. (a) Identification of cells that secrete IL-2 and IFN-c: PBMCs stimulated with PMA/ionomycin were subjectedto our secretion assay for IL-2 or IFN-c as described in the text, as well as standard intracellular staining, and a commercially available kit.Gates define fractions of cells positive for the secreted cytokine. Each graph depicts a single representative experiment performed on the

same set of cells. (b) Identification of cells that secrete other cytokines: PBMCs stimulated with PMA/ionomycin were subjected to a secre-

tion assay for MIP-1a, MIP-1b, and RANTES as described in the text. Gates define fractions of cells positive for the secreted cytokine. Eachgraph depicts a single representative experiment. (c) Use of quantum dots for the secretion assay: Quantum dots from Ocean Nanotech

Qdot 620 complexed with anti-CD45 cell targeting antibodies and anti-IL-2 antibodies were used in a cell secretion assay with PBMC stimu-

lated with PMA/ionomycin. Cells were also stained with an anti-CD45 antibody. IL-2 is detected in 11% of cells. The Qdots can also be used

to identify the targeted cells, all CD45 stained cells (dark density plot) are also stained by the Qdot IL-2/CD45 nanoparticle, compared to

nonstained cells (red density plot).

BRIEF REPORT


ACKNOWLEDGMENT

The authors thank L. Margolis for his support and stimu-

lating discussions.

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A universal nanoparticle cell secretion capture assay

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