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XX

Cytokine ImmunocytochemistryReagents and Techniques for Microscopic Analysis of Cytokine-Producing Cells

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XX XXi 1

Cover image is an artistic rendering of cytokine immunocytochemical staining and does not represent actual data.

All products are for research use only. Not for use in diagnostic or therapeutic procedures.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Immunocytochemical Measurements of Mouse Cytokines . . . . . . 2

Immunocytochemical Measurements of Human Cytokines . . . . . 3

Important Points to Consider when Choosing a Method to Study Cytokine Production by Individual Cells . . . . . . 4

Comparison of Cytokine Immunocytochemistry and FlowCytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Relative Sensitivities of Immunocytochemistry and Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Critical Parameters in Immunocytochemistry . . . . . . . . . . . . . . . 7

Schematic of Staining Protocol . . . . . . . . . . . . . . . . . . . . . . . . 8-9

Analysis of Mouse TNF-α-Producing Cells by Immunocytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

Features of Intracellular Staining . . . . . . . . . . . . . . . . . . . . . . . 11

Comparison of Immunocytochemical Detection Systems . . . . . . 11

Cytokine Immunocytochemistry Protocol . . . . . . . . . . . . . . . 12-13

Product Listing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Controls for Cytokine Immunocytochemistry . . . . . . . . . . . . . . 15

Cytokine Immunocytochemistry Program Overview . . . . . . . . . 16

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16

Table of Contents IntroductionThe term cytokines refers to a broad group of signaling proteins that, in general,are produced transiently after cellular activation, act locally as autocrine, juxtacrineor paracrine biological response modifiers, and exert their actions by binding tospecific high-affinity receptors on target cells. The vast cytokine group includesmembers of the interleukin, interferon, tumor necrosis factor, colony stimulatingfactor, transforming growth factor and chemokine families of proteins. Due to thecritical roles played by cytokines in the regulation of immunity and inflammation,there has been increased emphasis on defining the qualitative, quantitative,temporal and spatial patterns of their expression. These types of analyses havebenefited tremendously from the recent introduction of methods that allow for thecharacterization of cytokine-producing cells at the single cell level. Such methodsare often based on the use of monoclonal antibodies that are specific for a particularcytokine and immunofluorescent or immunoenzymatic techniques to detect andquantify specific antibody binding to target cytokines (Sander et al., 1991). In thelatter case, immunocytochemistry (ICC) can be a powerful immunoenzymatictechnique that allows for the detection of the cytokines associated with individualcells that have been immobilized on microscopic slides.

An important goal for PharMingen has been to develop and to provide highquality antibodies that can be used to study cytokine expression at the single celllevel. In this mailer, PharMingen is pleased to provide a list of selected antibodyclones that are now available for the immunocytochemical staining of humanand mouse cytokine-producing cells. Moreover, reagents and methods arepresented that are suitable for the immunoenzymatic analysis of cytokineprotein expression using the technique of immunocytochemistry.

We are thankful to Dr. Jan Andersson (Karolinska Institute, Sweden) and Dr. Thomas E. Fehniger (Astra Draco, Sweden) for consulting with PharMingen in thedevelopment of methods and reagents for the immunocytochemical analysis ofcytokine-producing cells.

Orders 1-800-848-MABS (6227) • Phone 619-812-8800 World Wide Web http://www.pharmingen.com

Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 World Wide Web http://www.pharmingen.com

XX XXi 1

Cover image is an artistic rendering of cytokine immunocytochemical staining and does not represent actual data.

All products are for research use only. Not for use in diagnostic or therapeutic procedures.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Immunocytochemical Measurements of Mouse Cytokines . . . . . . 2

Immunocytochemical Measurements of Human Cytokines . . . . . 3

Important Points to Consider when Choosing a Method to Study Cytokine Production by Individual Cells . . . . . . 4

Comparison of Cytokine Immunocytochemistry and FlowCytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Relative Sensitivities of Immunocytochemistry and Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Critical Parameters in Immunocytochemistry . . . . . . . . . . . . . . . 7

Schematic of Staining Protocol . . . . . . . . . . . . . . . . . . . . . . . . 8-9

Analysis of Mouse TNF-α-Producing Cells by Immunocytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

Features of Intracellular Staining . . . . . . . . . . . . . . . . . . . . . . . 11

Comparison of Immunocytochemical Detection Systems . . . . . . 11

Cytokine Immunocytochemistry Protocol . . . . . . . . . . . . . . . 12-13

Product Listing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Controls for Cytokine Immunocytochemistry . . . . . . . . . . . . . . 15

Cytokine Immunocytochemistry Program Overview . . . . . . . . . 16

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16

Table of Contents IntroductionThe term cytokines refers to a broad group of signaling proteins that, in general,are produced transiently after cellular activation, act locally as autocrine, juxtacrineor paracrine biological response modifiers, and exert their actions by binding tospecific high-affinity receptors on target cells. The vast cytokine group includesmembers of the interleukin, interferon, tumor necrosis factor, colony stimulatingfactor, transforming growth factor and chemokine families of proteins. Due to thecritical roles played by cytokines in the regulation of immunity and inflammation,there has been increased emphasis on defining the qualitative, quantitative,temporal and spatial patterns of their expression. These types of analyses havebenefited tremendously from the recent introduction of methods that allow for thecharacterization of cytokine-producing cells at the single cell level. Such methodsare often based on the use of monoclonal antibodies that are specific for a particularcytokine and immunofluorescent or immunoenzymatic techniques to detect andquantify specific antibody binding to target cytokines (Sander et al., 1991). In thelatter case, immunocytochemistry (ICC) can be a powerful immunoenzymatictechnique that allows for the detection of the cytokines associated with individualcells that have been immobilized on microscopic slides.

An important goal for PharMingen has been to develop and to provide highquality antibodies that can be used to study cytokine expression at the single celllevel. In this mailer, PharMingen is pleased to provide a list of selected antibodyclones that are now available for the immunocytochemical staining of humanand mouse cytokine-producing cells. Moreover, reagents and methods arepresented that are suitable for the immunoenzymatic analysis of cytokineprotein expression using the technique of immunocytochemistry.

We are thankful to Dr. Jan Andersson (Karolinska Institute, Sweden) and Dr. Thomas E. Fehniger (Astra Draco, Sweden) for consulting with PharMingen in thedevelopment of methods and reagents for the immunocytochemical analysis ofcytokine-producing cells.

Orders 1-800-848-MABS (6227) • Phone 619-812-8800 World Wide Web http://www.pharmingen.com

Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 World Wide Web http://www.pharmingen.com

2 3

Immunocytochemical Measurements of Mouse Cytokines Immunocytochemical Measurements of Human Cytokines

Figure 1. Immunocytochemical detection of cytokine-producing cells using PharMingen’s antibodies directed againstmouse and human cytokines.Mouse splenocytes were RBC-lysed, washed and cultured withPMA (5 ng/ml, Sigma) and ionomycin (500 ng/ml, Sigma) for 4 hrat 37°C with GolgiPlug™ (Cat. No. 2301KZ) that contains the pro-tein transport inhibitor, brefeldin A. Cells were harvested and stained by immunocytochemistry (ICC) for mouse IL-2 (JES6-1A12, A), IFN-γ (XMG1.2, B), and TNF-α (MP6-XT22, C).Alternatively, the RBC-lysed splenocytes were enriched for CD4+

cells and were cultured for 2 days with plate bound anti-mouseCD3 and soluble anti- mouse CD28 in the presence of recombinantIL-2 and recombinant IL-4. The cells were subsequently harvested,washed and recultured with recombinant IL-2 and recombinant IL-4 for an additional 3 days. Finally, the cells were harvested,washed and cultured (4 hr) with PMA and ionomycin in the pres-ence of GolgiPlug™ (Cat. No. 2301KZ). Cells were harvested andstained by ICC for mouse IL-3 (MP2-8F8, D), IL-4 (11B11, E), IL-10 (JES5-16E3, F) and GM-CSF (MP1-22E9, G).

Human PBMC were isolated by Lymphoprep (Nycomed) density gradient centrifugation and were stimulated with LPS (1 µg/ml, Sigma) overnight in the presence ofGolgiStop™ that contains the protein transport inhibitor, monensin (Cat. No. 2092KZ). Cells were stained for human IL-6 (MQ2-6A3, H). Alternatively human PBMCwere cultured for 2 days with plate bound anti-CD3 and soluble anti-mouse CD28 in the presence of recombinant IL-2 and recombinant IL-4. The cells were subse-quently harvested, washed and recultured with recombinant IL-2 and recombinant IL-4 for an additional 3 days. Finally, the cells were harvested, washed and cultured(4 hr) with PMA and ionomycin in the presence of GolgiStop™ (Cat. No. 2092KZ). Cells were harvested and stained by ICC for human IL-2 (MQ1-17H2, I), human IL-5 (JES1-39D10, J), human IL-13 (JES10-5A2, K), human GM-CSF (BVD2-21C11, L), and human TNF-α (MAB11, M). The specificity of staining was confirmed byusing the appropriate immunoglobulin isotype and ligand-blocking controls (A-M; Nomarski optics; original magnification 400 X).

(A) Mouse IL-2 (Clone JES6-1A12) (B) Mouse IFN-γ (Clone XMG1.2) (H) Human IL-6 (Clone MQ2-6A3) (I) Human IL-2 (Clone MQ1-17H2)

(C) Mouse TNF-α (Clone MP6-XT22) (D) Mouse IL-3 (Clone MP2-8F8)

(E) Mouse IL-4 (Clone 11B11) (F) Mouse IL-10 (Clone JES5-16E3)

(J) Human IL-5 (Clone JES1-39D10) (K) Human IL-13 (Clone JES10-5A2)

(L) Human GM-CSF (Clone BVD2-21C11) (M) Human TNF-α (Clone MAB11)

(G) Mouse GM-CSF (Clone MP1-22E9)

Orders 1-800-848-MABS (6227) • Phone 619-812-8800 World Wide Web http://www.pharmingen.com

Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 World Wide Web http://www.pharmingen.com

2 3

Immunocytochemical Measurements of Mouse Cytokines Immunocytochemical Measurements of Human Cytokines

Figure 1. Immunocytochemical detection of cytokine-producing cells using PharMingen’s antibodies directed againstmouse and human cytokines.Mouse splenocytes were RBC-lysed, washed and cultured withPMA (5 ng/ml, Sigma) and ionomycin (500 ng/ml, Sigma) for 4 hrat 37°C with GolgiPlug™ (Cat. No. 2301KZ) that contains the pro-tein transport inhibitor, brefeldin A. Cells were harvested and stained by immunocytochemistry (ICC) for mouse IL-2 (JES6-1A12, A), IFN-γ (XMG1.2, B), and TNF-α (MP6-XT22, C).Alternatively, the RBC-lysed splenocytes were enriched for CD4+

cells and were cultured for 2 days with plate bound anti-mouseCD3 and soluble anti- mouse CD28 in the presence of recombinantIL-2 and recombinant IL-4. The cells were subsequently harvested,washed and recultured with recombinant IL-2 and recombinant IL-4 for an additional 3 days. Finally, the cells were harvested,washed and cultured (4 hr) with PMA and ionomycin in the pres-ence of GolgiPlug™ (Cat. No. 2301KZ). Cells were harvested andstained by ICC for mouse IL-3 (MP2-8F8, D), IL-4 (11B11, E), IL-10 (JES5-16E3, F) and GM-CSF (MP1-22E9, G).

Human PBMC were isolated by Lymphoprep (Nycomed) density gradient centrifugation and were stimulated with LPS (1 µg/ml, Sigma) overnight in the presence ofGolgiStop™ that contains the protein transport inhibitor, monensin (Cat. No. 2092KZ). Cells were stained for human IL-6 (MQ2-6A3, H). Alternatively human PBMCwere cultured for 2 days with plate bound anti-CD3 and soluble anti-mouse CD28 in the presence of recombinant IL-2 and recombinant IL-4. The cells were subse-quently harvested, washed and recultured with recombinant IL-2 and recombinant IL-4 for an additional 3 days. Finally, the cells were harvested, washed and cultured(4 hr) with PMA and ionomycin in the presence of GolgiStop™ (Cat. No. 2092KZ). Cells were harvested and stained by ICC for human IL-2 (MQ1-17H2, I), human IL-5 (JES1-39D10, J), human IL-13 (JES10-5A2, K), human GM-CSF (BVD2-21C11, L), and human TNF-α (MAB11, M). The specificity of staining was confirmed byusing the appropriate immunoglobulin isotype and ligand-blocking controls (A-M; Nomarski optics; original magnification 400 X).

(A) Mouse IL-2 (Clone JES6-1A12) (B) Mouse IFN-γ (Clone XMG1.2) (H) Human IL-6 (Clone MQ2-6A3) (I) Human IL-2 (Clone MQ1-17H2)

(C) Mouse TNF-α (Clone MP6-XT22) (D) Mouse IL-3 (Clone MP2-8F8)

(E) Mouse IL-4 (Clone 11B11) (F) Mouse IL-10 (Clone JES5-16E3)

(J) Human IL-5 (Clone JES1-39D10) (K) Human IL-13 (Clone JES10-5A2)

(L) Human GM-CSF (Clone BVD2-21C11) (M) Human TNF-α (Clone MAB11)

(G) Mouse GM-CSF (Clone MP1-22E9)

Orders 1-800-848-MABS (6227) • Phone 619-812-8800 World Wide Web http://www.pharmingen.com

Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 World Wide Web http://www.pharmingen.com

XX XX4 5

Important points to consider whenchoosing a method to study cytokineproduction by individual cells.The method of choice for studying cytokine production at the single cell level isstrictly dependent upon the experimental model system being used, the availablefacilities and equipment, and the types of experimental questions that one seeks toanswer (see table). Presently, the most widely used method to characterize thenature of individual cytokine-producing cells has been immunofluorescent stainingcoupled with flow cytometric analysis. This constitutes a rapid and highly sensitivetechnique that offers the capacity for multiparameter characterization of individualcytokine-producing cells. A potential drawback of this technique is that it requiresaccess to a flow cytometer and a computer with appropriate software for dataacquisition and analysis. Immunocytochemistry, on the other hand, achieves asimilar sensitivity when compared with flow cytometry-based methods byemploying a straightforward enzyme-amplified detection system (Figure 2, page 6).As a result, immunocytochemical results can be acquired with the use of an ordinarylight microscope that is common to most research laboratories. In addition, theprocessed specimens can be stored without loss of quality and can be reexaminedas needed for an extended period of time. Moreover, because individual cellmorphology can be visualized by using this method, one can potentially identifydifferent cell types as well as distinguish between cytokine-producing cells and cellsthat stain positively due to receptor-mediated cytokine binding (Gordon, 1991).

An important consideration when performing immunocytochemical studies is thatthe number of cells that can be observed in each optical field of a microscopic slideis finite. Thus, in order to identify significant numbers of cytokine-producing cells,one may need to observe numerous optical fields. This may be problematic in caseswhere the number of cytokine-producing cells may be very low (less than 1%). Forexample, when the investigator is interested in studying antigen-specific responses,it may be difficult to count statistically-significant numbers of cytokine-positive cells(Suni et al. 1998). An additional consideration is that while flow cytometers routinelyquantify the signal, similar measurements with immunocytochemistry requireadditional image analysis equipment (Björk et al. 1996). Despite these concerns formany types of experimental systems, immunocytochemistry constitutes anextremely powerful technique for enumerating and characterizing cytokine-producing cells.

Comparison of Cytokine Immunocytochemistry and Flow Cytometry

CytokineImmunocytochemistry

Sensitivity

DetectionSystem

Features

Considerations

•High

•Enzyme-amplified antibody detection system

•Detects cytokine production at the single cell level

•Rapid assay•Simultaneous morphological and

cytokine staining analysis: enables distinction of intracellularversus cell surface- bound cytokines

•Processed samples can be stored for long periods without loss of quality

•Requires a light microscope for analysis

•Finite number of cells can be visualized in each optical field

•Image analysis equipment and appropriate software can be usedfor quantitation of cytokine staining signals

•High

•Fluorescent antibody-based Sensitive electronic detection and amplification of fluorescent antibody signals

•Detects cytokine production at the single cell level

•Rapid assay•Multiparameter analysis of

physical and fluorescent stainingcharacteristics of cytokine-producing cells

•Processed samples can be stored up to 1 wk at 4°C

•Requires a flow cytometer for analysis

•Large number of cells can be acquired and analyzed

•Flow cytometer enables quantitation of cytokine staining signals

CytokineFlow Cytometry

Orders 1-800-848-MABS (6227) • Phone 619-812-8800 World Wide Web http://www.pharmingen.com

Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 World Wide Web http://www.pharmingen.com

XX XX4 5

Important points to consider whenchoosing a method to study cytokineproduction by individual cells.The method of choice for studying cytokine production at the single cell level isstrictly dependent upon the experimental model system being used, the availablefacilities and equipment, and the types of experimental questions that one seeks toanswer (see table). Presently, the most widely used method to characterize thenature of individual cytokine-producing cells has been immunofluorescent stainingcoupled with flow cytometric analysis. This constitutes a rapid and highly sensitivetechnique that offers the capacity for multiparameter characterization of individualcytokine-producing cells. A potential drawback of this technique is that it requiresaccess to a flow cytometer and a computer with appropriate software for dataacquisition and analysis. Immunocytochemistry, on the other hand, achieves asimilar sensitivity when compared with flow cytometry-based methods byemploying a straightforward enzyme-amplified detection system (Figure 2, page 6).As a result, immunocytochemical results can be acquired with the use of an ordinarylight microscope that is common to most research laboratories. In addition, theprocessed specimens can be stored without loss of quality and can be reexaminedas needed for an extended period of time. Moreover, because individual cellmorphology can be visualized by using this method, one can potentially identifydifferent cell types as well as distinguish between cytokine-producing cells and cellsthat stain positively due to receptor-mediated cytokine binding (Gordon, 1991).

An important consideration when performing immunocytochemical studies is thatthe number of cells that can be observed in each optical field of a microscopic slideis finite. Thus, in order to identify significant numbers of cytokine-producing cells,one may need to observe numerous optical fields. This may be problematic in caseswhere the number of cytokine-producing cells may be very low (less than 1%). Forexample, when the investigator is interested in studying antigen-specific responses,it may be difficult to count statistically-significant numbers of cytokine-positive cells(Suni et al. 1998). An additional consideration is that while flow cytometers routinelyquantify the signal, similar measurements with immunocytochemistry requireadditional image analysis equipment (Björk et al. 1996). Despite these concerns formany types of experimental systems, immunocytochemistry constitutes anextremely powerful technique for enumerating and characterizing cytokine-producing cells.

Comparison of Cytokine Immunocytochemistry and Flow Cytometry

CytokineImmunocytochemistry

Sensitivity

DetectionSystem

Features

Considerations

•High

•Enzyme-amplified antibody detection system

•Detects cytokine production at the single cell level

•Rapid assay•Simultaneous morphological and

cytokine staining analysis: enables distinction of intracellularversus cell surface- bound cytokines

•Processed samples can be stored for long periods without loss of quality

•Requires a light microscope for analysis

•Finite number of cells can be visualized in each optical field

•Image analysis equipment and appropriate software can be usedfor quantitation of cytokine staining signals

•High

•Fluorescent antibody-based Sensitive electronic detection and amplification of fluorescent antibody signals

•Detects cytokine production at the single cell level

•Rapid assay•Multiparameter analysis of

physical and fluorescent stainingcharacteristics of cytokine-producing cells

•Processed samples can be stored up to 1 wk at 4°C

•Requires a flow cytometer for analysis

•Large number of cells can be acquired and analyzed

•Flow cytometer enables quantitation of cytokine staining signals

CytokineFlow Cytometry

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XX XX6 7

Critical Parameters inImmunocytochemistry1. Fixation: The proper fixation of cells is a very critical step for performing

immunocytochemical analyses. In this regard, formaldehyde has beenshown to be a gentle fixation agent that preserves cellular morphology andsize (and cytokine antigenicity) without cell loss or aggregation (Sander et al., 1991).

2. Permeabilization: Amongst a number of detergents and organic solventsused for cell permeabilization (including acetone, ethanol, methanol, n-octyl-beta-D-glucopyranoside, saponin and Triton-X), saponin was shownto be the best choice for intracellular cytokine staining (Sander et al., 1991,Björk., 1995). Saponin is a plant derivative that has high affinity forcholesterol. It is suggested that during permeabilization, saponinintercalates into the membrane and replaces cholesterol (Willingham &Pastan, 1985). Because this is a reversible process, it is important thatsaponin is constantly present in all staining buffers during incubations ofcells with antibodies (Willingham & Pastan, 1985; Sander et al., 1991).

3. Protein transport inhibitors: Immunocytochemistry can be used toexamine cells activated in the absence of protein transport inhibitors due tothe high sensitivity achieved with enzyme-amplified detection systems(Figure 3, page 10). This allows for cells to be stained ex vivo for intracellularcytokines without the need for in vitro restimulation. Alternatively, theinvestigator can apply protein transport inhibitors [e.g., GolgiStop™

containing monensin (Cat. No. 2092KZ) or GolgiPlug™ containingbrefeldin A (Cat. No. 2301KZ)] to cells during stimulatory cultures toaugment the detection of cytokine-producing cells caused by theintracellular accumulation of cytokine proteins.

4. Detection system: Like other immunoenzymatic techniques, the sensitivityof the immunocytochemical staining technique for detecting cytokine-producing cells is dependent upon the enzyme-substrate system used. Thepreferred enzyme for cytokine detection has been horseradish peroxidasebecause it gives strong signals and a non-diffuse staining pattern (Figure 4,page 11). For high-sensitivity detection of cytokine-producing cells withincell populations, the use of the Avidin:Biotinylated Complex procedure(ABC method) (Hsu et al., 1981a, Hsu et al., 1981b) is recommended. TheABC method employs biotinylated secondary antibodies and preformedAvidin:Biotinylated enzyme Complexes as a detection system. Thisprocedure appears to be very sensitive and to give low background stainingsignals because the amount of the primary antibody required for detectionof intracellular cytokines can be very small. As a result of our in-houseevaluations, we recommend an ICC detection method that utilizesAvidin:Biotinylated horseradish peroxidase complexes.

5. Cytokine specific antibodies: The choice of a particular cytokine-specificantibody is very critical. High affinity, monoclonal anti-cytokine antibodiesare recommended for immunocytochemical staining, as opposed topolyclonal antibody preparations, because of their capacity to targetstrong, highly-specific signals with correspondingly low backgroundstaining. In addition, the anti-cytokine antibody must have the capacity torecognize cytokine proteins after cell fixation with formaldehyde andpermeabilization with saponin. The same antibody clones presented in thismailer have not been evaluated in ICC using other methods.

12%

GM-CSF (PE)

Rel

ativ

e C

ell N

um

ber

77%

GM-CSF (PE)R

elat

ive

Cel

l Nu

mb

er

Figure 2. Detection of GM-CSF-producing cells by ICC and light microscopic analysis with a sensitivity comparable to that of immunofluorescent staining and flow cytometry. Human PBMC were isolated by Lymphoprep (Nycomed) density gradient centrifugation and were stimulated overnight with PMA andionomycin in the presence of GolgiStop™ that contains the protein transport inhibitor, monensin (Cat. No. 2092KZ). Cells were divided in two aliquots. The firstaliquot was stained for human GM-CSF (BVD2-21C11) by ICC and examined by light microscopy (A) and the second aliquot was tested by immunofluorescentstaining and flow cytometry (B). Alternatively human PBMC cells were cultured for 2 days with plate bound anti-CD3 and soluble anti-mouse CD28 in the presenceof recombinant IL-2 and recombinant IL-4. The cells were subsequently harvested, washed and recultured with recombinant IL-2 and recombinant IL-4 for an addi-tional 3 days. Finally, the cells were harvested, washed and cultured (4 hr) with PMA and ionomycin in the presence of GolgiStop™(Cat. No. 2092KZ). Cells were divided in two aliquots. The first aliquot was stained for human GM-CSF (BVD2-21C11) by ICC and examined by light microscopy (C)and the second aliquot was tested by immunofluorescent staining and flow cytometry (D), (A and C Nomarski optics; original magnification, 400 X). Specificity ofstaining was confirmed by using the appropriate immunoglobulin isotype and ligand-blocking controls. The two different activation protocols resulted in different lev-els of GM-CSF producing cells that were detectable with both flow cytometry and immunocytochemistry. The percentage of cytokine producing cells by ICC wasestimated by enumeration of at least 200 cells by light microscopy. The percentages of GM-CSF producing cells was 11% (A) and 70% (C).

(A) (B)

(C) (D)

Relative Sensitivities of Immunocytochemistry and Flow Cytometry

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XX XX6 7

Critical Parameters inImmunocytochemistry1. Fixation: The proper fixation of cells is a very critical step for performing

immunocytochemical analyses. In this regard, formaldehyde has beenshown to be a gentle fixation agent that preserves cellular morphology andsize (and cytokine antigenicity) without cell loss or aggregation (Sander et al., 1991).

2. Permeabilization: Amongst a number of detergents and organic solventsused for cell permeabilization (including acetone, ethanol, methanol, n-octyl-beta-D-glucopyranoside, saponin and Triton-X), saponin was shownto be the best choice for intracellular cytokine staining (Sander et al., 1991,Björk., 1995). Saponin is a plant derivative that has high affinity forcholesterol. It is suggested that during permeabilization, saponinintercalates into the membrane and replaces cholesterol (Willingham &Pastan, 1985). Because this is a reversible process, it is important thatsaponin is constantly present in all staining buffers during incubations ofcells with antibodies (Willingham & Pastan, 1985; Sander et al., 1991).

3. Protein transport inhibitors: Immunocytochemistry can be used toexamine cells activated in the absence of protein transport inhibitors due tothe high sensitivity achieved with enzyme-amplified detection systems(Figure 3, page 10). This allows for cells to be stained ex vivo for intracellularcytokines without the need for in vitro restimulation. Alternatively, theinvestigator can apply protein transport inhibitors [e.g., GolgiStop™

containing monensin (Cat. No. 2092KZ) or GolgiPlug™ containingbrefeldin A (Cat. No. 2301KZ)] to cells during stimulatory cultures toaugment the detection of cytokine-producing cells caused by theintracellular accumulation of cytokine proteins.

4. Detection system: Like other immunoenzymatic techniques, the sensitivityof the immunocytochemical staining technique for detecting cytokine-producing cells is dependent upon the enzyme-substrate system used. Thepreferred enzyme for cytokine detection has been horseradish peroxidasebecause it gives strong signals and a non-diffuse staining pattern (Figure 4,page 11). For high-sensitivity detection of cytokine-producing cells withincell populations, the use of the Avidin:Biotinylated Complex procedure(ABC method) (Hsu et al., 1981a, Hsu et al., 1981b) is recommended. TheABC method employs biotinylated secondary antibodies and preformedAvidin:Biotinylated enzyme Complexes as a detection system. Thisprocedure appears to be very sensitive and to give low background stainingsignals because the amount of the primary antibody required for detectionof intracellular cytokines can be very small. As a result of our in-houseevaluations, we recommend an ICC detection method that utilizesAvidin:Biotinylated horseradish peroxidase complexes.

5. Cytokine specific antibodies: The choice of a particular cytokine-specificantibody is very critical. High affinity, monoclonal anti-cytokine antibodiesare recommended for immunocytochemical staining, as opposed topolyclonal antibody preparations, because of their capacity to targetstrong, highly-specific signals with correspondingly low backgroundstaining. In addition, the anti-cytokine antibody must have the capacity torecognize cytokine proteins after cell fixation with formaldehyde andpermeabilization with saponin. The same antibody clones presented in thismailer have not been evaluated in ICC using other methods.

12%

GM-CSF (PE)

Rel

ativ

e C

ell N

um

ber

77%

GM-CSF (PE)

Rel

ativ

e C

ell N

um

ber

Figure 2. Detection of GM-CSF-producing cells by ICC and light microscopic analysis with a sensitivity comparable to that of immunofluorescent staining and flow cytometry. Human PBMC were isolated by Lymphoprep (Nycomed) density gradient centrifugation and were stimulated overnight with PMA andionomycin in the presence of GolgiStop™ that contains the protein transport inhibitor, monensin (Cat. No. 2092KZ). Cells were divided in two aliquots. The firstaliquot was stained for human GM-CSF (BVD2-21C11) by ICC and examined by light microscopy (A) and the second aliquot was tested by immunofluorescentstaining and flow cytometry (B). Alternatively human PBMC cells were cultured for 2 days with plate bound anti-CD3 and soluble anti-mouse CD28 in the presenceof recombinant IL-2 and recombinant IL-4. The cells were subsequently harvested, washed and recultured with recombinant IL-2 and recombinant IL-4 for an addi-tional 3 days. Finally, the cells were harvested, washed and cultured (4 hr) with PMA and ionomycin in the presence of GolgiStop™(Cat. No. 2092KZ). Cells were divided in two aliquots. The first aliquot was stained for human GM-CSF (BVD2-21C11) by ICC and examined by light microscopy (C)and the second aliquot was tested by immunofluorescent staining and flow cytometry (D), (A and C Nomarski optics; original magnification, 400 X). Specificity ofstaining was confirmed by using the appropriate immunoglobulin isotype and ligand-blocking controls. The two different activation protocols resulted in different lev-els of GM-CSF producing cells that were detectable with both flow cytometry and immunocytochemistry. The percentage of cytokine producing cells by ICC wasestimated by enumeration of at least 200 cells by light microscopy. The percentages of GM-CSF producing cells was 11% (A) and 70% (C).

(A) (B)

(C) (D)

Relative Sensitivities of Immunocytochemistry and Flow Cytometry

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XX XX8 9

1

2

4

5

3

Load cells on adhesionslides

Fix adherent cells

Block with 1% (w/v)BSA-PBS

Block with goat serumin buffer containingsaponin

Block endogenousperoxidase

20

15

30

10

-80˚C

Slides can be storedat -80˚C indefinitelybefore the stainingprocedure

W a s h 2 x

W a s h 2 x

W a s h 2 x

10

10

10

BSA

peroxidaseblock

endogenousperoxidase

Intracellular Cytokine Immunocytochemistry - Overview of Staining Protocol

30

W a s h 2 x10

saponin

Fc receptor

Goat Igblock

7

8

9

10

Incubate slides withprimary Ab

Add biotinylatedSecondary Ab

Add ABC solution

Add DAB substrate

30

30

60

W a s h 2 x

W a s h 2 x

W a s h 2 x10

10

10

cytokinespecificantibody

secondaryantibody

ABCcomplex

DABsubstrate

W a s h

Mount slides11

Examine slides by microscopy

6 Block endogenousbiotin

30

W a s h 2 x10

endogenousbiotin

biotinblock

cytokine

<5

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XX XX8 9

1

2

4

5

3

Load cells on adhesionslides

Fix adherent cells

Block with 1% (w/v)BSA-PBS

Block with goat serumin buffer containingsaponin

Block endogenousperoxidase

20

15

30

10

-80˚C

Slides can be storedat -80˚C indefinitelybefore the stainingprocedure

W a s h 2 x

W a s h 2 x

W a s h 2 x

10

10

10

BSA

peroxidaseblock

endogenousperoxidase

Intracellular Cytokine Immunocytochemistry - Overview of Staining Protocol

30

W a s h 2 x10

saponin

Fc receptor

Goat Igblock

7

8

9

10

Incubate slides withprimary Ab

Add biotinylatedSecondary Ab

Add ABC solution

Add DAB substrate

30

30

60

W a s h 2 x

W a s h 2 x

W a s h 2 x10

10

10

cytokinespecificantibody

secondaryantibody

ABCcomplex

DABsubstrate

W a s h

Mount slides11

Examine slides by microscopy

6 Block endogenousbiotin

30

W a s h 2 x10

endogenousbiotin

biotinblock

cytokine

<5

Orders 1-800-848-MABS (6227) • Phone 619-812-8800 World Wide Web http://www.pharmingen.com

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XX XX10 11

Figure 3. Immunocytochemical identification of TNF-α-producing cells in cultures with or without protein transport inhibitors. BALB/c splenocyteswere stimulated with PMA (5 ng/ml, Sigma) and ionomycin (500 ng/ml, Sigma) for 4 hr in the presence of GolgiStop™ (Cat. No. 2092KZ) (B), GolgiPlug™(Cat. No. 2301KZ) (A), or no protein transport inhibitor (C). Cells were stained with anti-mouse TNF-α (MP6-XT22) (A-C) or rat IgG1 (R3-34) (data not shown).Pre incubation of anti-mouse TNF-α antibody with purified recombinant mouse TNF-α protein (Cat. No 19321T) completely blocked the staining of MP6-XT22(D), (A-D; original magnification, 400 X).

Figure 4. Horseradish Peroxidase is the optimal detectionsystem for cytokine immunocytochemistry. Human PBMCwere isolated by Lymphoprep (Nycomed) density gradient cen-trifugation and were stimulated overnight with PMA and iono-mycin in the presence of GolgiStop™ (Cat. No. 2092KZ). Cellswere stained for human GM-CSF (BVD2-21C11) by ICC (see arrows) using an AKP- (A) or an HRP- (B) based detectionsystem. The specificity of staining was confirmed by using theappropriate immunoglobulin isotype and ligand blocking controls(A and B; Nomarski optics, original magnification, 400 X).

Features of Intracellular StainingThe majority of cytokines have N-terminal signal sequences that allow them tofollow the classical pathway of protein secretion. The classical pathway of proteinsecretion involves accumulation of the protein in the lumen of the endoplasmicreticulum and subsequent transfer to the Golgi compartment (Walter et al., 1994).The accumulation of cytokines in the Golgi prior to their secretion can be visualizedunder the microscope as perinuclear staining. Such staining is very characteristicfor the cytokine-producing cells and can be very easily distinguished fromextracellular binding to the plasma membrane (Figure 3). Members of theinterleukin-1 family, such as IL-1α and IL-1β, that lack N-terminal signal sequences,appear to follow an alternative pathway of secretion that does not involve theGolgi apparatus (Stevenson et al., 1992). These cells are characterized by a ratherdiffuse staining throughout the cytoplasm (Andersson, 1992; Björk, 1995).

Analysis of Mouse TNF-α-Producing Cells by Immunocytochemistry

Comparison of Immunocytochemical Detection Systems

(A) Brefeldin A (GolgiPlug™) (B) Monensin (GolgiStop™)

(C) No protein transport inhibitor (D) Ligand blocking control

(A) Alkaline Phosphatase (AKP)

(B) Horseradish Peroxidase (HRP)

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XX XX10 11

Figure 3. Immunocytochemical identification of TNF-α-producing cells in cultures with or without protein transport inhibitors. BALB/c splenocyteswere stimulated with PMA (5 ng/ml, Sigma) and ionomycin (500 ng/ml, Sigma) for 4 hr in the presence of GolgiStop™ (Cat. No. 2092KZ) (B), GolgiPlug™(Cat. No. 2301KZ) (A), or no protein transport inhibitor (C). Cells were stained with anti-mouse TNF-α (MP6-XT22) (A-C) or rat IgG1 (R3-34) (data not shown).Pre incubation of anti-mouse TNF-α antibody with purified recombinant mouse TNF-α protein (Cat. No 19321T) completely blocked the staining of MP6-XT22(D), (A-D; original magnification, 400 X).

Figure 4. Horseradish Peroxidase is the optimal detectionsystem for cytokine immunocytochemistry. Human PBMCwere isolated by Lymphoprep (Nycomed) density gradient cen-trifugation and were stimulated overnight with PMA and iono-mycin in the presence of GolgiStop™ (Cat. No. 2092KZ). Cellswere stained for human GM-CSF (BVD2-21C11) by ICC (see arrows) using an AKP- (A) or an HRP- (B) based detectionsystem. The specificity of staining was confirmed by using theappropriate immunoglobulin isotype and ligand blocking controls(A and B; Nomarski optics, original magnification, 400 X).

Features of Intracellular StainingThe majority of cytokines have N-terminal signal sequences that allow them tofollow the classical pathway of protein secretion. The classical pathway of proteinsecretion involves accumulation of the protein in the lumen of the endoplasmicreticulum and subsequent transfer to the Golgi compartment (Walter et al., 1994).The accumulation of cytokines in the Golgi prior to their secretion can be visualizedunder the microscope as perinuclear staining. Such staining is very characteristicfor the cytokine-producing cells and can be very easily distinguished fromextracellular binding to the plasma membrane (Figure 3). Members of theinterleukin-1 family, such as IL-1α and IL-1β, that lack N-terminal signal sequences,appear to follow an alternative pathway of secretion that does not involve theGolgi apparatus (Stevenson et al., 1992). These cells are characterized by a ratherdiffuse staining throughout the cytoplasm (Andersson, 1992; Björk, 1995).

Analysis of Mouse TNF-α-Producing Cells by Immunocytochemistry

Comparison of Immunocytochemical Detection Systems

(A) Brefeldin A (GolgiPlug™) (B) Monensin (GolgiStop™)

(C) No protein transport inhibitor (D) Ligand blocking control

(A) Alkaline Phosphatase (AKP)

(B) Horseradish Peroxidase (HRP)

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XX XX12 13

Reagents required1. Fixation Buffer: 5% formalin (10% formalin, CMS, Cat. No. 245-684)

is dissolved in phosphate buffered-saline (PBS) (Bacto® FA Buffer, Difco Laboratories, Cat. No. 2314-15-0).

2. Endogenous Peroxidase Blocking Buffer: DAKO Peroxidase BlockingReagent (DAKO, Cat. No. S2001).

3. Endogenous Biotin Blocking Buffer: Biotin/Avidin Blocking Kit (VectorLaboratories, Cat. No. SP-2001).

4. Antibody dilution buffer: PharMingen’s Cytokine IHC Diluent Buffersupplemented with saponin.

5. Microscopic slides: Adhesion Slides (Erie Scientific Company, Cat. No. ER-202B-AD) or for cytospins, Colorfrost®/Plus slides (Fisher, Cat. No.12-550-17).

6. Detection system: Vectastain® Elite ABC kit (Vector, Cat. No. PK-6100).

7. Mounting medium for short-term storage: Aqua-mount® (LernerLaboratories, Cat. No.13800).

Secondary antibodies 1. Biotin goat anti-rat IgG (PharMingen, Cat. No. 20392D)

2. Biotin goat anti-mouse IgG (PharMingen, please inquire)

Procedure for immunocytochemical staining of single-cell preparationsThis procedure describes the immunoenzymatic technique of staining cytokineswithin individual cells that are immobilized on microscopic slides via adherence(adherent slides) or centrifugation (cytospins).

Adhesion slides 1. Harvest cells and wash them twice in PBS using centrifugation (400 x g for

5 min) to remove residual protein.

2. Adjust the cell concentration at 4 x 106 to 5 x 106 cells/ml in PBS.

3. Place 20 µl of the cell suspension in each well of the adhesion slides andlet them adhere at room temperature (RT) for 20 min. Please note that theslides should be washed in PBS at RT for 5 min before transferring the cells.

4. Fix cells on slides using fixation buffer for 15 min at RT.

5. Wash slides 2X in PBS with 5 min incubations.

6. Block slides with PBS supplemented with 1% (w/v) BSA (Sigma, Cat. No. A43-78) for 30 min at RT or 10 min at 37°C.

7. Wash slides 2X in PBS and proceed with staining or air dry them and storethem at -80˚C for future use.

8. Incubate slides with 20 µl of 1% goat serum and PBS with 0.1% (w/v)saponin for 30 min at RT.

9. Wash slides 2X with PBS with 5 min incubations.

10. Block endogenous peroxidase activity with Endogenous PeroxidaseBlocking Buffer (20 µl/well) for 10 min at RT.

11. Wash 2X in PBS with 5 min incubations.

12. Incubate each well with Avidin (20 µl/well) for 15 min.

13. Wash 2X in PBS with 5 min incubations.

14. Incubate each well with Biotin (20 µl/well) for 15 min.

15. Wash 2X in PBS with 5 min incubations.

16. Incubate each well for 1 hr at RT with 20 µl of purified cytokine-specificantibody or appropriate immunoglobulin isotype control diluted inPharMingen’s Cytokine IHC Diluent Buffer supplemented with saponin.

17. Wash slides 2X in PBS with 5 min incubations.

18. Incubate each well with 20 µl of a biotinylated secondary antibody dilutedin IHC Cytokine Diluent Buffer for 30 min at RT.

19. Wash 2X in PBS with 5 min incubations.

20. Apply 20 µl of Vectastain Elite ABC solution (1:2 diluted) to each well onslides and incubate for 30 min at RT.

21. Wash slides 2X with PBS with 5 minutes incubations.

22. Incubate with 3-3´-Diaminobenzidine tetra hydrochloride (DAB), (Vector,Cat. No. SK4100) for less than 5 min at RT.

23. Stop the development of the color reaction by washing with PBS.

24. The slides are subsequently mounted in short-term storage mounding medium.

Cytospins 1. Assemble the Cytospin’s sample chamber (e.g. Cytospin 3, Shandon, UK or

comparable centrifuge), filter card, slide and cytospin racks according tomanufacturer’s specifications.

2. Load 40 µl of approximately 1 x 106 cells to each sample chamber.

3. Spin slides at 600 rpm for 2 min.

4. Take slides out of the cytospin rack and place them on a staining rack.

5. For fixation and staining please follow the steps 4 through 24 specifiedabove for staining cells on adhesion slides.

Note: PharMingen is currently developing an ICC Staining Kit that will include fixationbuffer, antibody diluent buffer, endogenous peroxidase blocking buffer and detectionsystem (please inquire).

Cytokine Immunocytochemistry Protocol

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XX XX12 13

Reagents required1. Fixation Buffer: 5% formalin (10% formalin, CMS, Cat. No. 245-684)

is dissolved in phosphate buffered-saline (PBS) (Bacto® FA Buffer, Difco Laboratories, Cat. No. 2314-15-0).

2. Endogenous Peroxidase Blocking Buffer: DAKO Peroxidase BlockingReagent (DAKO, Cat. No. S2001).

3. Endogenous Biotin Blocking Buffer: Biotin/Avidin Blocking Kit (VectorLaboratories, Cat. No. SP-2001).

4. Antibody dilution buffer: PharMingen’s Cytokine IHC Diluent Buffersupplemented with saponin.

5. Microscopic slides: Adhesion Slides (Erie Scientific Company, Cat. No. ER-202B-AD) or for cytospins, Colorfrost®/Plus slides (Fisher, Cat. No.12-550-17).

6. Detection system: Vectastain® Elite ABC kit (Vector, Cat. No. PK-6100).

7. Mounting medium for short-term storage: Aqua-mount® (LernerLaboratories, Cat. No.13800).

Secondary antibodies 1. Biotin goat anti-rat IgG (PharMingen, Cat. No. 20392D)

2. Biotin goat anti-mouse IgG (PharMingen, please inquire)

Procedure for immunocytochemical staining of single-cell preparationsThis procedure describes the immunoenzymatic technique of staining cytokineswithin individual cells that are immobilized on microscopic slides via adherence(adherent slides) or centrifugation (cytospins).

Adhesion slides 1. Harvest cells and wash them twice in PBS using centrifugation (400 x g for

5 min) to remove residual protein.

2. Adjust the cell concentration at 4 x 106 to 5 x 106 cells/ml in PBS.

3. Place 20 µl of the cell suspension in each well of the adhesion slides andlet them adhere at room temperature (RT) for 20 min. Please note that theslides should be washed in PBS at RT for 5 min before transferring the cells.

4. Fix cells on slides using fixation buffer for 15 min at RT.

5. Wash slides 2X in PBS with 5 min incubations.

6. Block slides with PBS supplemented with 1% (w/v) BSA (Sigma, Cat. No. A43-78) for 30 min at RT or 10 min at 37°C.

7. Wash slides 2X in PBS and proceed with staining or air dry them and storethem at -80˚C for future use.

8. Incubate slides with 20 µl of 1% goat serum and PBS with 0.1% (w/v)saponin for 30 min at RT.

9. Wash slides 2X with PBS with 5 min incubations.

10. Block endogenous peroxidase activity with Endogenous PeroxidaseBlocking Buffer (20 µl/well) for 10 min at RT.

11. Wash 2X in PBS with 5 min incubations.

12. Incubate each well with Avidin (20 µl/well) for 15 min.

13. Wash 2X in PBS with 5 min incubations.

14. Incubate each well with Biotin (20 µl/well) for 15 min.

15. Wash 2X in PBS with 5 min incubations.

16. Incubate each well for 1 hr at RT with 20 µl of purified cytokine-specificantibody or appropriate immunoglobulin isotype control diluted inPharMingen’s Cytokine IHC Diluent Buffer supplemented with saponin.

17. Wash slides 2X in PBS with 5 min incubations.

18. Incubate each well with 20 µl of a biotinylated secondary antibody dilutedin IHC Cytokine Diluent Buffer for 30 min at RT.

19. Wash 2X in PBS with 5 min incubations.

20. Apply 20 µl of Vectastain Elite ABC solution (1:2 diluted) to each well onslides and incubate for 30 min at RT.

21. Wash slides 2X with PBS with 5 minutes incubations.

22. Incubate with 3-3´-Diaminobenzidine tetra hydrochloride (DAB), (Vector,Cat. No. SK4100) for less than 5 min at RT.

23. Stop the development of the color reaction by washing with PBS.

24. The slides are subsequently mounted in short-term storage mounding medium.

Cytospins 1. Assemble the Cytospin’s sample chamber (e.g. Cytospin 3, Shandon, UK or

comparable centrifuge), filter card, slide and cytospin racks according tomanufacturer’s specifications.

2. Load 40 µl of approximately 1 x 106 cells to each sample chamber.

3. Spin slides at 600 rpm for 2 min.

4. Take slides out of the cytospin rack and place them on a staining rack.

5. For fixation and staining please follow the steps 4 through 24 specifiedabove for staining cells on adhesion slides.

Note: PharMingen is currently developing an ICC Staining Kit that will include fixationbuffer, antibody diluent buffer, endogenous peroxidase blocking buffer and detectionsystem (please inquire).

Cytokine Immunocytochemistry Protocol

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14 15

Controls for CytokineImmunocytochemistryPositive ControlsVarious in vitro methods have been reported for stimulating cytokine-producing cells. These methods include polyclonal activators such as phorbolesters plus calcium ionophore, phytohemaglutinin, Staphylococcus enterotoxinB, or monoclonal antibodies directed against subunits of the TCR/CD3complex (with or without antibodies directed against costimulatory receptors,such as, CD28). For details on cell stimulation please refer to PharMingen’sCytokine/Chemokine Applications Manual.

Negative controlsTo assure specificity of staining one or more of the following negative controlsare recommended:

• Immunoglobulin isotype controls: Stain cells using an antibody with the same isotype but with unrelated specificity as that of the cytokine-specific antibody.

• Ligand blocking control: Pre-block anti-cytokine antibodies with purified recombinant cytokine protein for 1 hr at RT. Note: Not all preparations of recombinant cytokine protein are useful for this control (e.g., mouse and human IFN-γ). The binding of antibody-cytokine complexes to cytokine receptors may be a factor in the poor blocking capacity of some recombinant cytokine proteins.

• Negative control: Stain cells that are not actively producing cytokines. For example, non-activated cells can be used as a negative control.

Anti-Cytokine Antibodies for Immunocytochemistry

Mouse

Specificity Clone Isotype Size Cat . No. Price ($)IL-2 JES6-1A12 rat IgG2a 0.25 mg 20191C 250 IL-3 MP2-8F8 rat IgG1 0.25 mg 20201C 250IL-4 11B11 rat IgG1 0.25 mg 20211C 250IL-10 JES5-16E3 rat IgG2b 0.25 mg 20221C 250TNF-α MP6-XT22 rat IgG1 0.25 mg 20231C 250IFN-γ XMG1.2 rat IgG1 0.25 mg 20241C 250GM-CSF MP1-22E9 rat IgG2a 0.25 mg 20251C 250

Human

Specificity Clone Isotype Size Cat . No. Price ($)IL-2 MQ1-17H12 rat IgG2a 0.25 mg 20281C 250IL-5 JES1-39D10 rat IgG2a 0.25 mg 20371C 250IL-6 MQ2-6A3 rat IgG2a 0.25 mg 20291C 250IL-10 JES3-19F1 rat IgG2a 0.25 mg 20381C 250IL-13 JES10-5A2 rat IgG1 0.25 mg 20301C 250GM-CSF BVD2-21C11 rat IgG2a 0.25 mg 20311C 250TNF-α MAB11 mouse IgG1 0.25 mg 20321C 250

Isotype controls

Isotype Clone Size Cat . No. Price ($)Rat IgG1 R3-34 0.25 mg 20331C 250Rat IgG2a R35-95 0.25 mg 20341C 250Rat IgG2b R35-38 0.25 mg 20361C 250Mouse IgG1 A112.2 0.25 mg 20351C 250

Secondary Antibodies

Specificity Size Cat . No. Price ($)Biotin Polyclonal Goat anti-rat IgG 0.5mg 20392D 250

Cytokine Ligand Blocking Controls

Mouse

Recombinant protein Size Cat. No. Price ($)L-2 10 µg 19211T 250IL-3 10 µg 19221T 210IL-4 5 µg 19231V 275IL-10 5 µg 19281V 295TNF-α 10 µg 19321T 250GM-CSF 10 µg 19291T 395

Human

Recombinant protein Size Cat. No. Price ($)IL-2 10 µg 19621T 195IL-5 5 µg 19651V 275IL-6 5 µg 19661V 185IL-10 5 µg 19701V 250IL-13 5 µg 19731V 250GM-CSF 5 µg 19741V 375TNF-α 10 µg 19761T 195

Orders 1-800-848-MABS (6227) • Phone 619-812-8800 World Wide Web http://www.pharmingen.com

Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 World Wide Web http://www.pharmingen.com

14 15

Controls for CytokineImmunocytochemistryPositive ControlsVarious in vitro methods have been reported for stimulating cytokine-producing cells. These methods include polyclonal activators such as phorbolesters plus calcium ionophore, phytohemaglutinin, Staphylococcus enterotoxinB, or monoclonal antibodies directed against subunits of the TCR/CD3complex (with or without antibodies directed against costimulatory receptors,such as, CD28). For details on cell stimulation please refer to PharMingen’sCytokine/Chemokine Applications Manual.

Negative controlsTo assure specificity of staining one or more of the following negative controlsare recommended:

• Immunoglobulin isotype controls: Stain cells using an antibody with the same isotype but with unrelated specificity as that of the cytokine-specific antibody.

• Ligand blocking control: Pre-block anti-cytokine antibodies with purified recombinant cytokine protein for 1 hr at RT. Note: Not all preparations of recombinant cytokine protein are useful for this control (e.g., mouse and human IFN-γ). The binding of antibody-cytokine complexes to cytokine receptors may be a factor in the poor blocking capacity of some recombinant cytokine proteins.

• Negative control: Stain cells that are not actively producing cytokines. For example, non-activated cells can be used as a negative control.

Anti-Cytokine Antibodies for Immunocytochemistry

Mouse

Specificity Clone Isotype Size Cat . No. Price ($)IL-2 JES6-1A12 rat IgG2a 0.25 mg 20191C 250 IL-3 MP2-8F8 rat IgG1 0.25 mg 20201C 250IL-4 11B11 rat IgG1 0.25 mg 20211C 250IL-10 JES5-16E3 rat IgG2b 0.25 mg 20221C 250TNF-α MP6-XT22 rat IgG1 0.25 mg 20231C 250IFN-γ XMG1.2 rat IgG1 0.25 mg 20241C 250GM-CSF MP1-22E9 rat IgG2a 0.25 mg 20251C 250

Human

Specificity Clone Isotype Size Cat . No. Price ($)IL-2 MQ1-17H12 rat IgG2a 0.25 mg 20281C 250IL-5 JES1-39D10 rat IgG2a 0.25 mg 20371C 250IL-6 MQ2-6A3 rat IgG2a 0.25 mg 20291C 250IL-10 JES3-19F1 rat IgG2a 0.25 mg 20381C 250IL-13 JES10-5A2 rat IgG1 0.25 mg 20301C 250GM-CSF BVD2-21C11 rat IgG2a 0.25 mg 20311C 250TNF-α MAB11 mouse IgG1 0.25 mg 20321C 250

Isotype controls

Isotype Clone Size Cat . No. Price ($)Rat IgG1 R3-34 0.25 mg 20331C 250Rat IgG2a R35-95 0.25 mg 20341C 250Rat IgG2b R35-38 0.25 mg 20361C 250Mouse IgG1 A112.2 0.25 mg 20351C 250

Secondary Antibodies

Specificity Size Cat . No. Price ($)Biotin Polyclonal Goat anti-rat IgG 0.5mg 20392D 250

Cytokine Ligand Blocking Controls

Mouse

Recombinant protein Size Cat. No. Price ($)L-2 10 µg 19211T 250IL-3 10 µg 19221T 210IL-4 5 µg 19231V 275IL-10 5 µg 19281V 295TNF-α 10 µg 19321T 250GM-CSF 10 µg 19291T 395

Human

Recombinant protein Size Cat. No. Price ($)IL-2 10 µg 19621T 195IL-5 5 µg 19651V 275IL-6 5 µg 19661V 185IL-10 5 µg 19701V 250IL-13 5 µg 19731V 250GM-CSF 5 µg 19741V 375TNF-α 10 µg 19761T 195

Orders 1-800-848-MABS (6227) • Phone 619-812-8800 World Wide Web http://www.pharmingen.com

16

PharMingen’s CytokineImmunocytochemistry andImmunohistochemistry ProgramPharMingen’s Cytokine Immunocytochemistry Program is a major new facet ofour continuing effort to develop reagents and techniques that allow for thesuccessful study of cytokine and chemokine networks at the cellular andmolecular level. PharMingen scientists are currently investigating the applicationof Immunohistochemical methods and reagents that can be used to detectcytokine-producing cells within tissue sections. The information that oneobtains by studying cytokine expression in situ in tissue sections is uniquebecause histological information is retained unlike techniques that analyze cellsin suspension. Immunohistochemical staining is a particularly powerfultechnique for examining cytokine-driven responses that can be localized withinspecific anatomical compartments of the body. For example, it is nowappreciated that in certain autoimmune, inflammatory or infectious diseases(e.g., rheumatoid arthritis and HIV infection) the representations of cytokine-producing cells at the local sites of autoimmune or inflammatory responses canbe very different from those found in peripheral blood samples from the sameindividual (Smeets et al., 1998, Andersson et al., 1998, Barnes et al., 1993,Sparrelid et al., 1997, Raqib et al., 1995). Thus, the ability to detect cellsproducing a particular pattern of cytokines in situ should provide important newinsights into the mechanisms of the cytokine network in health and disease.

References

1. Andersson, J., J. Abrams, L. Björk, K. Funa, M. Litton, K. Agren, and U. Andersson. 1994. Concomitant in vivo production of19 different cytokines in human tonsils. Immunol. 83:16-24.

2. Andersson, J., L. Björk, C. A. Dinarello, H. Towbin, and U. Andersson. 1992. Lipopolysaccharide induces human interleukin-1 receptor antagonist and interleukin-1 production in the same cell. Eur J Immunol 22:2617-2623.

3. Andersson, J.,T. E. Fehniger, B. K. Patterson, J. Pottage, M. Angoli, P. Jones, H. Behbahani and A. Landay. 1998. Earlyreduction of immune activation in lymphoid tissue following highly active HIV therapy. AIDS 12:F123-F129.

4. Barnes, P.F., S. Lu, J. S. Abrams, E. Wang, M. Yamamura, R. L. Modlin. 1993. Cytokine production at the site of disease inhuman tuberculosis. Infect Immun 61(8):3482-3489.

5. Björk, L. 1995. The characteristics of the intracellular cytokine pattern. In Development of an image analysis system for theevaluation of cytokine production. Thesis, Stockholm.

6. Björk, L., T. E. Fehniger, U. Andersson, and J. Andersson. 1996. Computerized assessment of production of multiple humancytokines at the single-cell level using image analysis. J Leukoc Biol 59:287-295.

7. Gordon, M. Y. 1991. Hemopoietic growth factors and receptors: bound and free. Cancer Cells 3:127-133.8. Hsu, S. M., L. Raine, and H. Fanger. 1981a. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase

techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577-580.9. Hsu, S. M., L. Raine, and H. Fanger. 1981b. A comparative study of the peroxidase-antiperoxidase method and an avidin-

biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am J Clin Pathol 75:734-738.10. Raqib, R., A. A. Lindberg, L. Bjork, P. K. Bardhan, B. Wretlind, U. Andersson, and J. Andersson. 1995. Down-regulation of

gamma interferon, tumor necrosis factor type I, interleukin 1 (IL-1) type I, IL-3, IL-4, and transforming growth factor beta typeI receptors at the local site during the acute phase of Shigella infection. Infect Immun 63:3079.

11. Sander, B., J. Andersson, and U. Andersson. 1991. Assessment of cytokines by immunofluorescence and theparaformaldehyde-saponin procedure. Immunol. Rev. 119:65-93.

12. Smeets,T. J., R. J. E. M Dolhain, A. M. Miltenburg, R. de Kuiper, F. C. Breedveld, P. P. Tak. 1998. Poor expression of T cell-derived cytokines and activation and proliferation markers in early rheumatoid synovial tissue. Clin Immunol Immunopathol88(1):84-90.

13. Sparrelid, E., D. Emanuel, T. Fehniger, U. Andersson, and J. Andersson. 1997. Interstitial pneumonitis in bone marrow transplantrecipients is associated with local production of TH2-type cytokines and lack of T cell-mediated cytotoxicity. Transplantation63:1782.

14. Stevenson, F. T., F. Torrano, R. M. Locksley, and D. H. Lovett. 1992. Interleukin 1: the patterns of translation and intracellulardistribution support alternative secretory mechanisms. J Cell Physiol 152:223-231.

15. Suni, M. A., L. J. Picker, and V. C. Maino. 1998. Detection of antigen-specific T cell cytokine expression in whole blood byflow cytometry. J Immunol Methods 212:89-98.

16. Walter, P., R. Gilmore, and G. Blobel. 1984. Protein translocation across the endoplasmic reticulum. Cell 38:5-8.17. Willingham, M.C. and I. Pastan. 1985. An atlas of immunofluorescence in cultured cells. Academic Press, Inc. London, p 5.

Currently Available:• Technical Data Sheets for all PharMingen products for easy downloading

• Complete catalog search of PharMingen’s 3,000 products

• Hot New Product Announcements

• Special offers for free promotional items, posters, etc.

• Online registration to join our catalog and electronic mailing lists

Upcoming Features for 1999:• Expanded product information sections

• Online ordering

• Customer education sections focusing on hot areas of research(including Apoptosis, Cytokines/Chemokines, etc.)

Orders 1-800-848-MABS (6227) • Phone 619-812-8800 World Wide Web http://www.pharmingen.com

16

PharMingen’s CytokineImmunocytochemistry andImmunohistochemistry ProgramPharMingen’s Cytokine Immunocytochemistry Program is a major new facet ofour continuing effort to develop reagents and techniques that allow for thesuccessful study of cytokine and chemokine networks at the cellular andmolecular level. PharMingen scientists are currently investigating the applicationof Immunohistochemical methods and reagents that can be used to detectcytokine-producing cells within tissue sections. The information that oneobtains by studying cytokine expression in situ in tissue sections is uniquebecause histological information is retained unlike techniques that analyze cellsin suspension. Immunohistochemical staining is a particularly powerfultechnique for examining cytokine-driven responses that can be localized withinspecific anatomical compartments of the body. For example, it is nowappreciated that in certain autoimmune, inflammatory or infectious diseases(e.g., rheumatoid arthritis and HIV infection) the representations of cytokine-producing cells at the local sites of autoimmune or inflammatory responses canbe very different from those found in peripheral blood samples from the sameindividual (Smeets et al., 1998, Andersson et al., 1998, Barnes et al., 1993,Sparrelid et al., 1997, Raqib et al., 1995). Thus, the ability to detect cellsproducing a particular pattern of cytokines in situ should provide important newinsights into the mechanisms of the cytokine network in health and disease.

References

1. Andersson, J., J. Abrams, L. Björk, K. Funa, M. Litton, K. Agren, and U. Andersson. 1994. Concomitant in vivo production of19 different cytokines in human tonsils. Immunol. 83:16-24.

2. Andersson, J., L. Björk, C. A. Dinarello, H. Towbin, and U. Andersson. 1992. Lipopolysaccharide induces human interleukin-1 receptor antagonist and interleukin-1 production in the same cell. Eur J Immunol 22:2617-2623.

3. Andersson, J.,T. E. Fehniger, B. K. Patterson, J. Pottage, M. Angoli, P. Jones, H. Behbahani and A. Landay. 1998. Earlyreduction of immune activation in lymphoid tissue following highly active HIV therapy. AIDS 12:F123-F129.

4. Barnes, P.F., S. Lu, J. S. Abrams, E. Wang, M. Yamamura, R. L. Modlin. 1993. Cytokine production at the site of disease inhuman tuberculosis. Infect Immun 61(8):3482-3489.

5. Björk, L. 1995. The characteristics of the intracellular cytokine pattern. In Development of an image analysis system for theevaluation of cytokine production. Thesis, Stockholm.

6. Björk, L., T. E. Fehniger, U. Andersson, and J. Andersson. 1996. Computerized assessment of production of multiple humancytokines at the single-cell level using image analysis. J Leukoc Biol 59:287-295.

7. Gordon, M. Y. 1991. Hemopoietic growth factors and receptors: bound and free. Cancer Cells 3:127-133.8. Hsu, S. M., L. Raine, and H. Fanger. 1981a. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase

techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577-580.9. Hsu, S. M., L. Raine, and H. Fanger. 1981b. A comparative study of the peroxidase-antiperoxidase method and an avidin-

biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am J Clin Pathol 75:734-738.10. Raqib, R., A. A. Lindberg, L. Bjork, P. K. Bardhan, B. Wretlind, U. Andersson, and J. Andersson. 1995. Down-regulation of

gamma interferon, tumor necrosis factor type I, interleukin 1 (IL-1) type I, IL-3, IL-4, and transforming growth factor beta typeI receptors at the local site during the acute phase of Shigella infection. Infect Immun 63:3079.

11. Sander, B., J. Andersson, and U. Andersson. 1991. Assessment of cytokines by immunofluorescence and theparaformaldehyde-saponin procedure. Immunol. Rev. 119:65-93.

12. Smeets,T. J., R. J. E. M Dolhain, A. M. Miltenburg, R. de Kuiper, F. C. Breedveld, P. P. Tak. 1998. Poor expression of T cell-derived cytokines and activation and proliferation markers in early rheumatoid synovial tissue. Clin Immunol Immunopathol88(1):84-90.

13. Sparrelid, E., D. Emanuel, T. Fehniger, U. Andersson, and J. Andersson. 1997. Interstitial pneumonitis in bone marrow transplantrecipients is associated with local production of TH2-type cytokines and lack of T cell-mediated cytotoxicity. Transplantation63:1782.

14. Stevenson, F. T., F. Torrano, R. M. Locksley, and D. H. Lovett. 1992. Interleukin 1: the patterns of translation and intracellulardistribution support alternative secretory mechanisms. J Cell Physiol 152:223-231.

15. Suni, M. A., L. J. Picker, and V. C. Maino. 1998. Detection of antigen-specific T cell cytokine expression in whole blood byflow cytometry. J Immunol Methods 212:89-98.

16. Walter, P., R. Gilmore, and G. Blobel. 1984. Protein translocation across the endoplasmic reticulum. Cell 38:5-8.17. Willingham, M.C. and I. Pastan. 1985. An atlas of immunofluorescence in cultured cells. Academic Press, Inc. London, p 5.

Currently Available:• Technical Data Sheets for all PharMingen products for easy downloading

• Complete catalog search of PharMingen’s 3,000 products

• Hot New Product Announcements

• Special offers for free promotional items, posters, etc.

• Online registration to join our catalog and electronic mailing lists

Upcoming Features for 1999:• Expanded product information sections

• Online ordering

• Customer education sections focusing on hot areas of research(including Apoptosis, Cytokines/Chemokines, etc.)

Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 World Wide Web http://www.pharmingen.com

XX

Cytokine ImmunocytochemistryReagents and Techniques for Microscopic Analysis of Cytokine-Producing Cells

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