UNIT 14.21Measurement of Type I InterferonProduction

Rosalind E. Seeds1,2 and Joanna L. Miller2

1Sir William Dunn School of Pathology, University of Oxford, United Kingdom2Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, UnitedKingdom

ABSTRACT

The Basic Protocol in this unit describes measurement of murine interferon (IFN)α/β byintracellular staining for these cytokines and detection by flow cytometry. Alternate proto-cols detail an enzyme-linked immunoabsorbent assay (ELISA) for IFNα and a biologicalassay to measure IFN. The FACS assay allows measurement of IFNα/β production bydefined cell populations, while ELISA measures secreted IFNα. The bioassay measuresfunctional antiviral activity and is the most sensitive of the assays discussed. Theseassays therefore provide complementary methods to assess IFN production by murinecells. Curr. Protoc. Immunol. 92:14.21.1-14.21.11. C© 2011 by John Wiley & Sons, Inc.

Keywords: intracellular cytokine staining � ELISA � bioassay � type I interferon �

cytokines

INTRODUCTION

There are two distinct classes of interferon (IFN), type I and type II. Many cell typescan produce type I IFN (namely, IFNα and IFNβ), and this response can be elicited bya wide variety of stimuli, including factors from the host, viruses, and bacteria. Type IIIFN, IFNγ, is produced by natural killer (NK) cells and activated T cells (Boehm et al.,1997). Although IFNγ has antiviral functions, it also has distinct effects from type I IFNs.This unit illustrates techniques to measure type I IFN production. Type I IFN are keyimmune cytokines, playing a pivotal role in defense against viral infection, among otherfunctions, and the IFN assays detailed will enable the study of a number of aspects of thisresponse. Type I IFNs are rapidly induced during innate immune responses to infection,and they ultimately provide signals for initiation of the adaptive immune response.

The methods described in this unit are based on measurement of type I IFN production bymurine cells. In contrast, UNIT 6.9 provides methods for measurement of antiviral activityinduced by IFNα and IFNβ as well as type II IFN, IFNγ. In this unit, murine splenocytesstimulated with inactivated influenza virus or CpG oligodeoxynucleotide (ODN) areused to generate type I IFN. Three approaches for measuring the IFN produced bythese cells are included. In the Basic Protocol, a flow cytometric method (intracellularstaining) is used to identify IFNα/β production by the plasmacytoid dendritic cell (pDC)subset within splenocytes. In Alternate Protocol 1, an ELISA is used to measure IFNα

secretion. Finally, Alternate Protocol 2 describes a bioassay to measure functional anti-viral activity. UNIT 6.9 also includes a functional assay for type I IFN and describes an assayfor murine IFN-induced antiviral activity that employs vesicular stomatitis virus (VSV)and IFN-sensitive fibroblasts. Here, Alternate Protocol 2 measures antiviral activity ina test sample by its ability to protect L929 cells against cocal virus-induced cytopathiceffect (CPE).

These three complimentary assays are highly versatile and can be applied to measureIFN following a range of stimulations.

Current Protocols in Immunology 14.21.1-14.21.11, February 2011Published online February 2011 in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/0471142735.im1421s92Copyright C© 2011 John Wiley & Sons, Inc.

Innate Immunity

14.21.1

Supplement 92

Measurement ofType I Interferon

Production

14.21.2

Supplement 92 Current Protocols in Immunology

BASICPROTOCOL

INTRACELLULAR STAINING FOR IFNαα AND IFNββ

The flow cytometric analysis of IFNα/β measures intracellular IFN protein levels. Ex-amples of cells expressing IFNα/β are either spleen cells stimulated with inactivatedinfluenza virus or CpG ODN for 9 hr or bone marrow–derived macrophages stimulatedfor 24 hr with polyinosinic acid:polycytidylic acid (polyI:C). After 5 hr of stimulation,brefeldin A is added to prevent cytokine secretion from the cells. At the end of the stim-ulation, cells are fixed, permeabilized, and stained with antibodies against IFNα, IFNβ,and in the case of splenocytes, pDCs, to identify the IFNα/β producing population.

Materials

Single cell suspension of splenocytes (prepared as described in Seeds et al., 2009)RPMI medium (see recipe for serum-free RPMI) supplemented with 10% (v/v)

heat-inactivated fetal bovine serumCpG ODN 2216 (InvivoGen) or HAU β-propiolactone or ultraviolet-inactivated

influenza virusGolgiPlug or Brefeldin A (BD Biosciences)PBS (APPENDIX 2A)1% paraformaldehyde in PBSFACS block solution (see recipe)Anti-pDC monoclonal antibody (mAb) such as 120G8 (Asselin-Paturel et al.,

2003) directly labeled with FITC or Alexafluor488 (IMGENEX)Biotinylated anti-IFNα mAb, such as clones 4EA1 (Kawade and Watanabe, 1987)

or RMMA-1 (PBL InterferonSource)Biotinylated anti-IFNβ mAb, such as clone 7FD3 (Kawade and Watanabe, 1987)

or RMMB-1 (PBL Interferon source)Appropriate isotype control mAbFACS wash solution (see recipe), ice coldStreptavidin allophycocyanin (SA-APC) or other appropriate secondary detection

reagent

Class II biological safety cabinet6- or 24-well flat-bottomed tissue culture plates1.5-ml microcentrifuge tubes96-well V-bottomed plates37◦C, 5% CO2 humidified incubatorCell scrapers (BD Falcon)Refrigerated centrifugeFlow cytometer with minimum ability to measure two colors

1. In a class II biological safety cabinet, stimulate a single cell suspension of 2 × 107

spleen cells in 0.75 or 2 ml RPMI with serum in 24- or 6-well tissue culture plates,respectively, for 9 hr in a 37◦C, 5% CO2 humidified incubator with 1 μM CpG ODN2216 or 400 HAU β-propiolactone- or UV-irradiation–inactivated influenza virus.

Store spleen cells on ice until used, CpG ODN at −20◦C, and inactivated influenza virusat 4◦C. Work under sterile conditions in a class II biological safety cabinet. Differentstimuli may require optimization of the incubation time to generate detectable IFNα/β. Atime course of 6 to 24 hr could be tested.

Use inactivated virus to stimulate IFNα/β production by pDC alone and replicating virusesto stimulate IFN production by a range of cell types.

To detect IFNα/β production by a different cell population or using different markers,substitute the appropriate antibodies for 120G8. Similarly, other anti-IFNα/β antibodiescan be tested and optimized for use.

Innate Immunity

14.21.3

Current Protocols in Immunology Supplement 92

2a. After 5 hr, add 1 μl GolgiPlug per milliliter of medium and continue incubation.At the end of the stimulation, use a cell scraper to detach the cells from the wells.Remove the cells and medium and place in a 1.5-ml microcentrifuge tube. Centrifugethe cells 5 min at 350 × g, 4◦C. Discard the supernatant and resuspend the cell pelletby gently pipetting up and down in 1 ml PBS. Repeat three times and then resuspendthe cells in 100 μl of 1% paraformaldehyde and transfer to 96-well V-bottomed plates.Incubate for at least 1 hr at 4◦C.

Fixed samples should be stored up to 24 hr at 4◦C until assayed.

2b. Do not perform step 2a when cells are stimulated with the aim of measuring secretedcytokines (as described in Alternate Protocols 1 and 2). Instead, at the end of theincubation, collect the cell supernatant, centrifuge for 5 min at 350 × g, 4◦C, to pelletany cells. Transfer the supernatants to fresh tubes and store up to 1 year at −20◦C.

Freeze-thawing reduces IFN activity; therefore, cell supernatants should be frozen andstored in multiple aliquots in preparation for measurement of IFN by bioassay.

3. Stain samples of 1 × 106 cells in 50 μl FACS block for 30 min on ice to block mAbbinding to Fc receptors and minimize non-specific staining.

4. Stain cells in 100 μl total volume with 10 μg/ml 120G8-AlexaFluor488 to identifypDCs, when needed, and with 10 μg/ml 4EA1-biotin (anti-IFNα) or 7FD3-biotin(anti-IFNβ) mAb diluted in the FACS block solution for 1 hr on ice. As controls, alsostain cells with isotype matched control mAb for 120G8 and the IFNα/β mAb.

Antibody stocks should be stored at −20◦C and a working aliquot at 4◦C.

5. Wash cells three times with 50 μl cold FACS wash by centrifuging plate for 5 min at350 × g, 4◦C, to pellet any cells.

6. Incubate cells for 30 min on ice with 100 μl of 2 μg/ml SA-APC secondary detectionreagent diluted in FACS wash.

Store SA-APC according to manufacturer’s instruction at 4◦C.

7. Wash cells three times in 50 μl cold FACS wash (as in step 5) and then fix in 100 μlof 1% paraformaldehyde 30 min to 24 hr at 4◦C.

8. Analyze antibody staining on a flow cytometer. For example, initially analyze cellsby forward scatter and side scatter characteristics and gate out debris. Subsequentlyidentify the population of interest, e.g., pDCs, through specific antibody staining incomparison to isotype control staining. To quantify IFNα/β production, set a gate tocontain 1% to 5% of pDCs (120G8+ cells) from splenocytes stimulated with mediumalone. Apply the same gate to stimulated samples and compare the percent of cellsthat fall within this gate.

ALTERNATEPROTOCOL 1

MEASUREMENT OF IFNαα BY ELISA

ELISA can be used to measure IFNα secreted into the supernatant by cells stimulated asdescribed earlier, without the addition of brefeldin A. The IFNα ELISA differs from theflow cytometry assay discussed in the Basic Protocol as the ELISA measures secretedprotein, not protein accumulated within the cell. In addition, this IFNα ELISA is moresensitive than the intracellular staining protocol (Seeds et al., 2009); therefore, it candetect lower levels of IFNα. However, the ELISA has the disadvantage of not identifyingthe IFN-producing cell, which may be relevant when a mixed cell population is used.ELISA kits to measure IFNα are commercially available; however, it is often moreeconomical to optimize a protocol independently.

Measurement ofType I Interferon

Production

14.21.4

Supplement 92 Current Protocols in Immunology

Additional Materials (also see Basic Protocol)

ELISA wash solution (see recipe)ELISA blocking solution (see recipe)Murine IFNα standard (PBL InterferonSource)Rabbit polyclonal Ab against IFNα (PBL InterferonSource)Alkaline phosphatase–conjugated goat anti-rabbit IgGAlkaline buffer: 50 mM Na2CO3, 1 mM Mg2Cl2.6H2O, pH 9.8 in waterp-Nitrophenyl phosphate (Sigma)

96-well ELISA plates37◦C incubatorSpectrofluorometer (405-nm absorbance reading)

1. Incubate 96-well ELISA plates overnight at 4◦C with 50 μl/well of 4 μg/ml 4EA1or RMMA-1 (anti-IFNα) mAb to coat the wells with the capture Ab. Alternatively,coat plates for 2 hr at 37◦C.

This assay could be adapted for detection of IFNβ by using capture and/or detectionantibodies specific for IFNβ.

2. On the next day, wash the wells with 50 μl/well of ELISA wash solution added using amultichannel pipettor and then removing the liquid by vigorously inverting the plate.

3. Incubate wells with 200 μl/well ELISA blocking solution for 2 hr at 37◦C.

Cover the ELISA plate to prevent evaporation of the blocking solution during the incuba-tion. Prepare sample dilutions during this incubation to save time.

4. Remove the ELISA blocking solution and add 35 μl/well of sample or murine IFNα

standard, serially diluted two-fold (start IFNα standard dilutions from 40,000 U/ml) induplicate wells. In addition, add PBS alone to two wells as a negative control. Incubate2 hr at room temperature. Wash the plate three times in ELISA wash solution.

The IFNα standard should be stored as small aliquots at −80◦C until needed to reducefreeze-thawing, which lowers titer. Test samples should be stored at −20◦C or −80◦C.

5. Add 50 μl/well 100 U/ml rabbit polyclonal Ab against IFNα diluted in ELISAblocking solution to detect IFNα, and incubate 1 hr at room temperature. Then, washwells three times with 50 μl/well PBS.

6. Incubate for 1 hr at room temperature with 50 μl/well alkaline phosphatase–conjugated goat anti-rabbit IgG diluted 1/1000 in PBS.

7. Wash wells three times with 50 μl/well PBS and then three times with 50 μl/wellalkaline buffer.

8. Dissolve 1 tablet of p-nitrophenyl phosphate in 5 ml alkaline buffer and add 50 μl/well.

p-Nitrophenyl phosphate tablets should be stored at −20◦C.

9. Incubate the plate 10 min to 1 hr at room temperature and, when a strong color isvisible, read absorbance at 405 nm on a spectrofluorometer.

If the color is developing slowly, cover the samples and incubate at 37◦C to increasethe rate of color development. Steps 6 to 9 are to detect the rabbit polyclonal anti-IFNα

antibody.

10. Compare the mean absorbance of the duplicate samples to those of a standard curveof the IFNα to estimate the amount of IFNα in the samples.

A laboratory IFNα/β standard can be prepared, calibrated against the National Institutesof Health murine IFN-α standard, and used routinely to convert levels of IFN measuredto International Units (IU).

Innate Immunity

14.21.5

Current Protocols in Immunology Supplement 92

ALTERNATEPROTOCOL 2

MEASUREMENT OF IFN BY BIOASSAY

This IFN bioassay is a highly-sensitive alternative to the flow cytometric and ELISAmethods previously discussed. It uses relatively inexpensive reagents and equipmentto measure functional protein levels as the readout is antiviral activity, i.e., this assaymeasures the ability of the test sample to protect L929 cells against cocal virus-inducedcytopathic effect (CPE). A variety of cell (murine, human, or bovine fibroblasts) andchallenge virus (Semliki Forest virus or vesicular stomatitis virus) combinations havebeen utilized with most laboratories reading the endpoint as the dilution providing 50%protection to the cell monolayer. Every plate includes a virus control well, representingfull destruction (cells that receive only serum-free medium prior to infection with virus),and a cell control exhibiting no CPE (untreated and uninfected cells in serum-freemedium). One disadvantage of the bioassay is that the basic assay does not differentiatebetween detection of type I and type II IFN. This can be overcome through the use ofneutralizing concentrations of anti-IFNγ or anti-IFNα/β antibodies (described in step 5).The bioassay takes 5 to 6 days, so pre-planning is recommended.

Additional Materials (also see Basic Protocol)

L929 murine fibroblast cells (ATCC #CCL-1)Serum-free RPMICocal virus of quantified titer (ATCC #VR-435)5 mg/ml thiazolyl blue tetrazolium bromide (MTT)10% SDS, 0.1 % HCl in waterNeutralizing anti-IFNγ Ab such as clone R4-6A2 (BD Biosciences) or clone

HB170 (Havell, 1986)Neutralizing anti-IFNα/β Ab such as a combination of polyclonal anti-IFNβ Ab

and polyclonal anti-IFNα Ab (PBL InterferonSource)IFNγ standard (PBL InterferonSource)

96-well flat-bottomed and U-bottomed tissue culture platesSpectrofluorometer (555 nm and 680 nm)

1. Day 1: Seed L929 cells overnight in 100 μl serum-free RPMI in 96-well flat-bottomedtissue culture plates so that the monolayer is confluent the next morning.

Optimization of the cell number may be necessary depending on the growth rate of thecells used, but ∼4 × 104 cells/well should be 100% confluent the next day.

2. Day 2: Incubate the monolayers of L929 cells overnight at 37◦C with duplicate two-fold serial dilutions of either an IFNα standard (from 250 U/ml) or samples, dilutedin 100 μl serum-free RPMI in 96-well U-bottomed plates. Include wells on each platefor cell and virus controls (see step 3a below), which receive only 100 μl serum-freeRPMI on day 2.

The IFNα standard should be stored as aliquots at −80◦C until needed. Serial dilutionscan be prepared in sterile 96-well U-bottomed plates, using a multichannel pipettor, andthen transferred onto L929 cell monolayers. Care should be taken to add the samplesto the wells gently, so as not to disturb the cell monolayers. However, the authors havefound that removal of the supernatants from the L929 monolayers can be performed byvigorously inverting the whole plate.

3. Day 3: Challenge cells with a dose of cocal virus, diluted in 100 μl per well serum-free RPMI, which allows a range of CPE to be seen across the standard curve from100% viable cells at the highest IFNα levels to totally dead cells at the lower dosesof IFNα. Incubate for 2 days, or until there is 100% CPE in the virus control well(see below).

Measurement ofType I Interferon

Production

14.21.6

Supplement 92 Current Protocols in Immunology

The dose of cocal virus may need to be optimized, but a starting amount of 250 to2500 pfu/ml cocal virus is recommended (Symons et al., 1995). Cocal virus should bestored in aliquots at −80◦C.

As controls, on each plate incubate at least one well of cells with 100μl serum free RPMIalone (cell control) and one without IFN, but with cocal virus only (virus control). Whenthe assay is read, the cell control should be 100% alive and the virus control 100%dead.

4. Day 5: Determine the dilution that causes a 50% reduction in CPE, using one of thefollowing methods:

a. Add 10 μl of 5 mg/ml MTT to each well and incubate for 6 to 8 hr at 37◦C.Solubilize cells by adding 100 μl/well of 10% SDS/0.1% HCl in water andincubate overnight. Read the absorbance at the reference wavelength of 680 nmand test wavelength of 555 nm the following day.

CAUTION: MTT is a carcinogen and teratogen. Use appropriate precautions asadvised by the supplier. MTT should be stored at 4◦C in the dark.

b. Alternatively, determine the 50% CPE point for the standard curve and serialdilutions of samples microscopically, with reference to the appearance of cells inthe cell and virus control wells. When cells are dead, they detach from the surfaceand become round in appearance.

5. To determine whether type I or type II IFN is present in samples:

Antibodies should be specific and neutralizing for either type I or type II IFN. In addition,to determine the relative levels of IFNα and IFNβ subtypes in a mixed sample of type IIFN, a modification of the Basic Protocol could be used. The sample would need to beassayed for IFN while including a neutralizing concentration of either an anti-IFNα oranti-IFNβ Ab.

a. In preparation: Estimate the titer of IFN in the sample by bioassay (see steps 1 to4, above).

One aliquot of the sample can be used here to estimate the IFN titer and a secondaliquot used for the identification of the presence of type I or II IFN (see step 5c,below).

b. Day 1: Seed L929 cells so as to be confluent the following day as in step 1.

c. Day 2: Add 100 μl per well of the following samples (in duplicate) diluted inserum-free RPMI, to the L929 cells:i. Sample diluted to an IFN titer approximately two- to four-fold above the assaydetection limit (sample IFN titer calculated in step 5a), e.g., 4 to 125 U/ml, mixedwith serial two-fold dilutions of either anti-IFNγ mAb or anti-IFNα/β Ab startingat a neutralizing concentration (which can be calculated from the neutralizing unittiter given by the manufacturer).

It can be helpful to prepare the serial dilutions of anti-IFN Ab in sterile 96-well U-bottomed plates; using a multichannel pipettor, add an equal titer of IFN (of samplebeing characterized) to all these wells and then transfer100 μl/well onto the L929cell monolayers.

ii. To confirm that the neutralizing anti-IFN Ab are effective, include IFNα/βand -γ standards, mixed with serial two-fold dilutions of anti-IFNα/β mAb oranti-IFNγ Ab, respectively.iii. Duplicate wells of the highest concentration of the anti-IFNγ and anti-IFNα/βantibodies. Adding the Ab alone (with no IFN) will verify that the Ab is notprotective against virus infection, which would complicate the interpretation ofthe assay.

Innate Immunity

14.21.7

Current Protocols in Immunology Supplement 92

iv. In addition, reconfirm the titer of IFN in the sample as part of this experiment,i.e., duplicate two-fold serial dilutions of sample dilution prepared in step i. Alsoinclude virus and cell controls.

It is likely to be necessary to titrate samples and/or antibodies to optimize this assay.While time consuming (IFN titers in samples must initially be estimated followed byinhibition with either anti-IFNαβ or anti-IFNγ antibodies in a second bioassay),determination of the type of IFN produced in a particular cell-virus system needs onlyto be established for this system, not re-confirmed for every experiment.

d. Continue assay as in steps 3 and 4.

REAGENTS AND SOLUTIONSUse deionized, distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2A; for suppliers, see APPENDIX 5.

ELISA blocking solution

Prepare 10% (v/v) heat-inactivated fetal bovine serum in PBS (APPENDIX 2A). Store1 year at room temperature.

ELISA wash solution

Prepare 0.05% (v/v) Tween in PBS (APPENDIX 2A). Store 1 year at room temperature.

FACS block solution

FACS wash solution (see recipe) supplemented with:5% (v/v) rabbit serum4 μg/ml 2.4 G2 anti-Fcγ receptor mAb (BD Biosciences)Prepare fresh and store at 4◦C

FACS wash solution

0.5% (w/v) bovine serum albumin5 mM EDTA (APPENDIX 2A)2 mM NaN3

0.2% (w/v) saponin

The basic FACS wash solution (without saponin) can be stored up to 1 year at 4◦C, butsolution with saponin must be prepared fresh for each experiment.

Serum-free RPMI

RPMI supplemented with:20 U/ml penicillin0.02 mg/ml streptomycin2 mM L-glutamineStore 6 months at 4◦C

COMMENTARY

Background InformationType I IFN was discovered by Isaacs and

Lindenmann (1957) over 50 years ago due toits antiviral activity against influenza virus.Since then work has developed to characterizethe IFN family, study how IFN production isinduced, and elucidate the effects of IFN. Mea-surement of IFN production therefore enablesthese functional studies; studying immune re-sponses and cellular function.

There are four main types of assay usedto measure IFNα/β production. PCR is com-monly used to detect IFNα and IFNβ mRNAexpression by pure cell populations such asmacrophage or fibroblast cultures. ELISAsand bioassays can be used to measure se-creted IFNα/β. Finally, intracellular cytokinestaining protocols detect IFNα/β productionby flow cytometry. These assays have differ-ent advantages and disadvantages. Detection

Measurement ofType I Interferon

Production

14.21.8

Supplement 92 Current Protocols in Immunology

of mRNA levels by PCR will not identifythe IFN-producing cell unless a pure popu-lation is used and the results may not re-flect protein production. However, several sub-types of IFNα production can be assayedfor simultaneously. ELISAs on cell super-natants allow unambiguous detection of se-creted protein. IFN bioassays are longer, morecomplex experiments, but use less expen-sive reagents/equipment and detect productionof functional IFN proteins using a biologi-cally relevant assay system (protection of cellsfrom viral infection). Intracellular staining forIFNα/β production is a quick method for de-tecting the production of specific types of IFNprotein by identified cell types within a mixedcell population. Intracellular cytokine assayshave now been developed to detect IFNα pro-duction by murine, human, and porcine cells.For example, intracellular assays can detectIFNα production by murine bone marrow-derived DCs stimulated with polyI:C (Dieboldet al., 2003) and by bone marrow–derived Flt3ligand pDCs stimulated with inactivated in-fluenza virus (Diebold et al., 2004). Similarly,intracellular staining can detect IFNα/β pro-duction by spleen pDCs from mice infectedwith MCMV or stimulated with CpG ODN(Sjolin et al., 2006). These techniques, and oth-ers (Asselin-Paturel et al., 2005; Chan et al.,2006), to detect murine IFNα/β use the mono-clonal antibodies RMMA-1 (PBL Interferon-Source), F18 (Hycult Biotech), or RMMB-1(PBL InterferonSource) for cytokine staining.Although this unit has detailed methods forIFNα/β measurement, the basic principles ofthe intracellular staining and ELISA protocolscan be extended to the measurement of differ-ent cytokines, through selection of differentantibodies.

Critical ParametersThe level of type I IFN that is produced

will be the critical determinant of which assayis suitable. In the authors’ hands, the bioas-say is the most sensitive assay, followed byELISA, and then the intracellular staining pro-tocol (Seeds et al., 2009). The level of IFNproduced will depend on the dose and typeof stimulus, the cell types used, the strain ofmouse, and the length of stimulation. For ex-ample, splenocytes from the 129/SvEv mousestrain produced higher levels of IFNα/β thanthose from C57BL/6J mice, following virus orCpG stimulations.

If live virus is used for induction of IFNin the samples, then it will be necessary to

inactivate/remove this from cell culture super-natants before measuring IFN levels by bioas-say. Virus should be inactivated using a suit-able method that does not affect the activityof the IFN present in the sample, for exam-ple, treatment with β-propiolactone, or in thecase of influenza virus, 30-min adsorption onchicken red blood cells on ice.

TroubleshootingSee Table 14.21.1 for troubleshooting sug-

gestions.

Anticipated ResultspDC comprise ∼1% to 2% of the total

murine spleen cell population and should beclearly identifiable using either the 120G8mAb as described here or other pDC-specific mAbs. In addition, staining forCD11c and B220 will identify pDCs asthe B220+CD11cintermediate population. Figure14.21.1 shows an example of double stainingsplenocytes with 120G8 to identify pDCs andwith an anti-IFNα mAb. The spleen cells werestimulated for 9 hr with 20 or 200 HAU/ml in-activated influenza virus. When incubated withmedium alone, pDCs are identified as the dis-crete population in the top left of the dot plot(highlighted by the gate in Fig. 14.21.1). Fol-lowing stimulation, IFNα-positive pDCs ap-pear in the upper right area. Figure 14.21.2illustrates the measurement of IFNα pro-duction by ELISA by bone marrow–derivedmacrophages following stimulation for 24 hrwith polyI:C. Figure 14.21.3 shows IFN titersdetermined by bioassay following stimulationof C57BL/6J and MyD88 knock-out spleencells with inactivated influenza virus for 23 hr.

As the assays described here measure bi-ological immune responses, care should betaken to maintain consistent stimulation andassay conditions to minimize biological vari-ation and achieve the most consistent mea-surements. For example, precautions could in-clude using cells prepared from mice of thesame strain, sex, and approximate age. Par-ticular care should be taken when performingthe bioassay to measure IFN titers, as this as-say itself is also dependent on biological fac-tors, which can introduce additional variation.Quantification of results in the bioassay maybe affected by factors such as the growth con-ditions of the cells before use or the age of thestock cells at the time of use. These factors areeliminated by comparisons of titers with stan-dard IFN preparations. Either a laboratory orcommercial standard (calibrated with the NIH

14.21.9

Current Protocols in Immunology Supplement 92

Table 14.21.1 Troubleshooting

Problem Possible cause Solution

No IFNα/β detected by flowcytometry

Not enough IFNα/β produced tobe detectable

Alter dose of stimulus or length of time ofstimulation. Try more sensitive methods ofdetection such as ELISA or bioassay

IFN production may vary with mouse strain andcell type

Purify IFN producing cells to increaseconcentration

Optimize staining protocol withwell-characterized stimulus, e.g., CpG ODN

In the bioassay, the viruschallenge killseverything/nothing

Titer of virus too high/low Titrate the virus dose. Optimize so that when noIFN is added the virus kills all the cells, whilecells are 100 % viable when co-treated with thehighest IFN dose.

IFN concentration in samples istoo low

Stimulate cells in a smaller volume to increaseIFN concentration in samples

Some cells are still viable in thevirus control well on day 5 of thebioassay

Insufficient time for virus to killcells

Incubate cells for another 1/2 day to see if viruskilling progresses

Insufficient virus dose Titrate virus so that a dose is found which kills allof the cells in the virus control wells in thestandard time

Insufficient color develops inELISA for IFNα standard

Development reactionprogressing too slowly

Speed up reaction by incubation at 37◦C ratherthan at room temperature

Standard curve starts at too low adose

Start standard curve titration at higherconcentration

Sources of IFNα/β antibodies Limited resources and changes insuppliers

The availability of anti-IFNα/β antibodies mayvary therefore it could be necessary to source andtest the staining of available antibodies on areliable source of IFNα/β producing cells, suchas the virally stimulated splenocytes

medium only 20 HAU/ml 200 HAU/ml 2000 HAU/ml

IFNα1 10 100 1000

120G

B

1

10

100

1000

1 10 100 1000 1 10 100 1000 1 10 100 1000

Figure 14.21.1 Dose response of IFNα production by pDCs in response to inactivated influenza virus. A concentrationof 1 × 107 spleen cells/ml were incubated in a volume of 2 ml per condition in medium alone or with 20 to 2000 HAU/mlinactivated influenza virus (A/Guangdong/25/93), as indicated, for 9 hr. After 5 hr of stimulation, GolgiPlug was addedand the incubations continued. After a total of 9 hr, cells were fixed in 1% paraformaldehyde, permeabilized, and stainedwith 120G8 mAb to identify pDCs and RMMA-1 mAb to detect IFNα (highlighted by gate).

Measurement ofType I Interferon

Production

14.21.10

Supplement 92 Current Protocols in Immunology

25,000

20,000

15,000

10,000

5000

medium0

0.1 μg/mlpolyl:C

1 μg/mlpolyl:C

10 μg/mlpolyl:C

Stimulation

IFα

(U/m

l)

Figure 14.21.2 Bone marrow–derived macrophages produce IFNα upon polyI:C stimulation. Aconcentration of 2.5 × 106 C57BL/6J bone marrow–derived macrophages/ml was stimulated withthe indicated amounts of polyI:C or medium alone for 24 hr in a volume of 200 μl/stimulation. IFNα

production in the supernatants was assayed by ELISA in duplicate.

Strain

C57BL/6J MyD88 K/O

*p <0.01120,000

100,000

80,000

60,000

20,000

40,000

0

IFN

(U

/ml)

Figure 14.21.3 IFN production in response to influenza virus by C57BL/6J and MyD88 knock-outspleen cells. A concentration of 2.67 × 107 C57BL/6J or MyD88 knock-out spleen cells/ml wasstimulated for 23 hr in a volume of 0.75 ml/treatment with 533 HAU/ml inactivated influenza virus(A/Guangdong/25/93). Supernatants were collected and assayed in duplicate for IFN productionby bioassay. IFN production following stimulation with medium alone was always <270 U/ml (datanot shown). p-value was calculated by pared t-test.

Murine IFN-α Reference Reagent) should betitrated in each assay. Variation between plateswithin an assay can be assessed by comparingthe controls on each plate, which is regularlyvaried by an absorbance of <0.1, across upto 20 plates. The authors recommend assay-ing triplicate samples in duplicate for IFN toallow for statistical calculations. Even follow-ing normalization of IFN titer with referenceto a standard, calculated IFN titers for a sam-

ple may vary two- to four-fold between repeatexperiments. This level of variations seems in-herent in the bioassay, although relative levelsof IFN between different treatments, for ex-ample, remain the same.

Time ConsiderationsThe flow cytometric staining protocol for

measuring IFNα/β production is the quick-est of the three assays. This would typically

Innate Immunity

14.21.11

Current Protocols in Immunology Supplement 92

take 3.5 hr to complete, with several 30-min to1-hr incubation steps interspersed with shortpractical times required to add antibody and∼20 min per wash step. The mAb incubationsteps could be lengthened if needed. In addi-tion, once the cells are fully stained, they canbe left for 1 day at 4◦C before analysis. Withpractice, two or more 96-well plates can be as-sayed in 1 day. The ELISA will take ∼1 work-ing day to complete, again with lots of incu-bation steps. It is possible, when experienced,to assay several 96-well plates in 1 day. Thebioassay will take 5 or 6 days depending on theread-out method used: on day 1, L929 cells areplated, on day 2, IFN or sample is added, onday 3, cells are challenged with virus, on day5, CPE can be read microscopically or cellstreated with MTT to be analyzed on the fol-lowing day. The timing by day should not bealtered; however, there is flexibility to conducteach stage at a different time each day, e.g.,on day 1 evening, plate the cells, on day 2morning, add samples, on day 3 mid-day, addvirus. Many plates can be assayed in one ex-periment; however, the size of the experimentrequires 24 wells per sample; therefore, scaleup accordingly. With experience ten or more96-well plates (39 samples and the IFN stan-dard curve) can be assayed in one experiment.This becomes challenging if cell viability is as-sayed microscopically rather than by using theMTT reaction, as it will take several minutesto analyze each sample by eye.

Literature CitedAsselin-Paturel, C., Brizard, G., Pin, J.J., Briere,

F., and Trinchieri, G. 2003. Mouse strain dif-ferences in plasmacytoid dendritic cell fre-quency and function revealed by a novelmonoclonal antibody. J. Immunol. 171:6466-6477.

Asselin-Paturel, C., Brizard, G., Chemin, K.,Boonstra, A., O’Garra, A., Vicari, A., andTrinchieri, G. 2005. Type I interferon depen-dence of plasmacytoid dendritic cell activationand migration. J. Exp. Med. 201:1157-1167.

Boehm, U., Klamp, T., Groot, M., and Howard, J.C.1997. Cellular responses to interferon-gamma.Annu. Rev. Immunol. 15:749-795.

Chan, C.W., Crafton, E., Fan, H.N., Flook,J., Yoshimura, K., Skarica, M., Brockstedt,D., Dubensky, T.W., Stins, M.F., Lanier,

L.L., Pardoll, D.M., and Housseau, F. 2006.Interferon-producing killer dendritic cells pro-vide a link between innate and adaptive immu-nity. Nat. Med. 12:207-213.

Diebold, S.S., Montoya, M., Unger, H.,Alexopoulou, L., Roy, P., Haswell, L.E.,Al-Shamkhani, A., Flavell, R., Borrow, P., andReis e Sousa, C. 2003. Viral infection switchesnon-plasmacytoid dendritic cells into highinterferon producers. Nature 424:324-328.

Diebold, S.S., Kaisho, T., Hemmi, H., Akira, S.,and Reis e Sousa, C. 2004. Innate antiviral re-sponses by means of TLR7-mediated recogni-tion of single-stranded RNA. Science 303:1529-1531.

Havell, E.A. 1986. Purification and further charac-terization of an anti-murine interferon-gammamonoclonal neutralizing antibody. J. Interferon.Res. 6:489-497.

Isaacs, A. and Lindenmann, J. 1957. Virus interfer-ence. I. The interferon. Proc. R. Soc. Lond. BBiol. Sci. 147:258-267.

Kawade, T. and Watanabe, Y. 1987. Characteri-zation of rat monoclonal antibodies to mouseinterferon-α and -β. In Biology of the Inter-feron System pp. 197-201. Proceedings of theThird International TNO Meeting on the Biol-ogy of the Interferon System. The Hague, TheNetherlands.

Seeds, R.E., Gordon, S., and Miller, J.L. 2009.Characterization of myeloid receptor expressionand interferon alpha/beta production in murineplasmacytoid dendritic cells by flow cytomtery.J. Immunol. Methods 350:106-117.

Sjolin, H., Robbins, S.H., Bessou, G., Hidmark,A., Tomasello, E., Johansson, M., Hall, H.,Charifi, F., Karlsson Hedestam, G.B., Biron,C.A., Karre, K., Hoglund, P., Vivier, E., andDalod, M. 2006. DAP12 signaling regulatesplasmacytoid dendritic cell homeostasis anddown-modulates their function during viral in-fection. J. Immunol. 177:2908-2916.

Symons, J.A., Alcami, A., and Smith, G.L. 1995.Vaccinia virus encodes a soluble type I in-terferon receptor of novel structure and broadspecies specificity. Cell 81:551-560.

Key ReferencesPerler, L., Pfister, H., Schweizer, M., Peterhans,

E., and Jungi, T.W. 1999. A bioassay for inter-feron type I based on inhibition of Sendai virusgrowth. J. Immunol. Methods 222:189-196.

Vogel, S.N., Friedman, R.M., and Hogan, M.M.2001. Measurement of antiviral activity inducedby interferons alpha, beta, and gamma. Curr.Protoc. Immunol. 37:6.9.1-6.9.8.

Other helpful IFN bioassay protocols.