Flow cytometric measurement of intracellular cytokines

doi:10.1016/S0022-1759(00)00230-1

Journal of Immunological Methods

Volume 243, Issues 1–2, 21 September 2000, Pages 107-124

https://doi.org/10.1016/S0022-1759(00)00230-1 Get rights and content

Abstract

The identification of distinct T helper lymphocyte subsets (Th1/2) with polarised cytokine production has opened up new fields in immunobiology. Of the several alternative methods of monitoring cytokine production, flow cytometric analysis of intracellular staining has distinct advantages and pitfalls. It allows high throughput of samples and multiparameter characterisation of cytokine production on a single cell basis without the need for prolonged in vitro culture and cloning. However, these methods may cause important changes in cell surface phenotype which can make interpretation difficult.

Section snippets

Background

Throughout the field of immunology, flow cytometry remains a defining technology. While some surface markers correlate with function, inferring function from surface staining remains an inexact art. Cells with similar surface phenotype may synthesise different products and have different functional characteristics.

The identification of functional subsets of CD4⁺ T lymphocytes with polarised cytokine production was based originally on the characterisation of T cell clones using cytokine ELISA of

Basic materials

•
PMA (phorbol 12-myristate 13-acetate Sigma P-8139)
•
Ionomycin (Sigma I-0634)
•
Calcium ionophore A23187 (500 ng/ml, Sigma C-9275)
•
Brefeldin A (Sigma B-7651)
•
Monensin (Sigma M-5273)
•
Formaldehyde (Analar grade, 37–40%)
•
Saponin (Sigma S-7900)
•
DMSO (dimethyl-sulfoxide Sigma D-5879)
•
Recombinant cytokines (Pharmingen and various other suppliers)
•
FCS or BSA (Sigma A-7906)
•
DNAse I

Stock reagents and buffers

•
‘PMA and ionomycin’ stock is made up in DMSO at 10 and 100 μg/ml, respectively, stored at −80°C. Thawed aliquots must not be re-used.
•

Effect of different stimuli on the cytokine response of T cells

Resting cells do not normally make cytokines, so ICCS requires cells to be activated. Depending on the cytokine, levels peak some hours after stimulation then decrease within a few more hours or days (Openshaw et al., 1995). However, specific antigen, polyclonal mitogen and pharmacological activation may trigger different activation pathways. The investigator therefore needs to test whether a non-specific stimulus is equivalent to physiological activation for the purposes of the experiment.

Discussion

The widespread use of flow cytometry has fundamentally affected the way we view the structure and lineage of the cells that comprise the immune system. This revolution, based on surface markers, is now being followed by further refinements based on staining for internal antigens. Among these, intracellular cytokine staining has played an important part in defining functional subsets in mixed T cell populations. It allows both the frequency of cells producing specific cytokines to be estimated

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Flow cytometric measurement of intracellular cytokines

Abstract

Section snippets

Background

Basic materials

Stock reagents and buffers

Effect of different stimuli on the cytokine response of T cells

Discussion

Blood

Cell Immunol.

Clin. Immunol Immunopathol.

Blood

Blood

Lancet

Clin. Immunol. Immunopathol.

J. Immunol. Methods

J. Immunol. Methods

J. Allergy Clin. Immunol.

J. Immunol. Methods

J. Immunol. Methods

J. Allergy Clin. Immunol

Blood

Hum. Immunol

Exp. Cell Res.

J. Immunol. Methods

J. Biol. Chem.

J. Invest. Dermatol.

J. Immunol. Methods

Am. J. Pathol.

Am. J. Surg.

Concomitant in vivo production of 19 different cytokines in human tonsils

Immunology

Opposite role for interleukin-4 and interferon-gamma on CD30 and lymphocyte activation gene-3 (LAG-3) expression by activated naive T cells

Eur. J. Immunol.

Specific expression of surface interferon-gamma on interferon-gamma producing T cells from mouse and man

Eur. J. Immunol.

Interferon-gamma secretion by peripheral blood T-cell subsets in multiple sclerosis: correlation with disease phase and interferon-beta therapy

Ann. Neurol.

Increased intracellular Th1 cytokines in scid mice with inflammatory bowel disease

Eur. J. Immunol

Cytotoxic T cells clear virus but augment lung pathology in mice infected with respiratory syncytial virus

J. Exp. Med.

T-lymphocyte functionality assessed by analysis of cytokine receptor expression, intracellular cytokine expression, and femtomolar detection of cytokine secretion by quantitative flow cytometry

Cytometry

Detection of altered T helper 1 and T helper 2 cytokine production by peripheral blood mononuclear cells in patients with multiple sclerosis utilizing intracellular cytokine detection by flow cytometry and surface marker analysis

Clin. Diagn. Lab. Immunol

Differences in the frequency of cytokine-producing cells in antigenemic and nonantigenemic individuals with bancroftian filariasis

Infect. Immun.

Golgi-disturbing agents

Histochem. Cell Biol.

Determination of cytokine co-expression in individual splenic CD4+ and CD8+ T cells from influenza virus-immune mice

Immunology

Type 1 and type 2 cytokine-producing mouse CD4+ and CD8+ T cells in acute Schistosoma mansoni infection

Eur. J. Immunol

Intracellular cytokine expression in whole blood preparations from normals and patients with atopic dermatitis

Clin. Exp. Immunol

Single-cell cytokine analysis of gamma delta T cell responses to nonpeptide mycobacterial antigens

J. Immunol.

FACS-based isolation of slowly growing cells: double encapsulation of yeast in gel microdrops

Nat. Biotechnol.

T helper 1 response is dominant and localised to the synovial fluid in patients with Lyme arthritis

J. Immunol.

CD3-CD4-CD8-CD56-CD19-CD14− cells and CD3+CD8 dull-positive cells produce IL-4 in AIDS patients

Br. J. Haematol.

Intracellular interferon-gamma expression in natural killer cells precedes lung CD8+ T cell recruitment during respiratory syncytial virus infection

J. Gen. Virol.

Th1 and Th2 cytokine induction in pulmonary T-cells during infection with respiratory syncytial virus

J. Gen. Virol.

IL-12 treatment attenuates Th2 and B cell responses but does not improve vaccine-enhanced lung illness

J. Immunol.

CD8+ T-cells control Th2-driven pathology during pulmonary respiratory syncytial virus infection

Eur. J. Immunol.

Host genetic determinants of vaccine-induced eosinophilia during respiratory syncytial virus infection

J. Immunol.

The specific cytotoxic T-cell response of adult volunteers to infection with respiratory syncytial virus

Immun. Infect. Dis.

Changes in intracellular cytokine levels in newborn and adult lymphocytes induced by HSV-1

J. Med. Virol.

A comparison of the performance in vitro of precision cut tissue slices and suspensions of human spleen with special reference to immunoglobulin and cytokine production

Hum. Antibodies Hybridomas

Determination of cytokine co-expression in individual splenic CD4⁺ and CD8⁺ T cells from influenza virus-immune mice

Type 1 and type 2 cytokine-producing mouse CD4⁺ and CD8⁺ T cells in acute Schistosoma mansoni infection

Intracellular interferon-gamma expression in natural killer cells precedes lung CD8⁺ T cell recruitment during respiratory syncytial virus infection

CD8⁺ T-cells control Th2-driven pathology during pulmonary respiratory syncytial virus infection

Interleukin-13 is produced by activated human CD45RA⁺ and CD45RO⁺ T cells: modulation by interleukin-4 and interleukin-12