Detection of intracellular interferon-gamma by light microscopy using an immunoperoxidase technique: correlation with the corresponding mRNA and protein product.
Identifying individual cytokine-producing cells may help to acquire insight into immunological processes. This study was designed to adapt a technique for the detection of individual cytokine-producing cells from an immunofluorescence to an immunoperoxidase staining procedure. The production of interferon-gamma (IFN-gamma) by anti-CD3-activated cloned human T cells was used as a model system. After the conditions for the staining procedure were optimized, the immunoperoxidase technique was slightly more sensitive than the immunofluorescence technique. The intracellular staining for IFN-gamma was preceded or paralleled by IFN-gamma mRNA production and followed by accumulation of IFN-gamma in the supernatant. It is concluded that intracellular IFN-gamma can easily be detected using an immunoperoxidase procedure. This procedure is highly sensitive and allows quantification of the production of multiple cytokines by counting the percentage of positively staining cells.
Dolhain
R J
RJ
Department of Rheumatology, University Hospital, Leiden, The Netherlands.
Andersson
U
U
ter Haar
N T
NT
Brinkman
B M
BM
Verweij
C L
CL
Daha
M R
MR
Breedveld
F C
FC
Miltenburg
A M
AM
D016207:Cytokines / Q000235:genetics; D005455:Fluorescent Antibody Technique; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D007371:Interferon-gamma / Q000032:analysis* / Q000235:genetics; D007375:Interleukin-1 / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism; D007377:Interleukin-3 / Q000378:metabolism; D015847:Interleukin-4 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D017472:Receptors, Interleukin-1 / Q000037:antagonists & inhibitors*; D013194:Staining and Labeling; D013601:T-Lymphocytes / Q000737:chemistry*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D016207:Cytokines; D007375:Interleukin-1; D007376:Interleukin-2; D007377:Interleukin-3; D015850:Interleukin-6; D016209:Interleukin-8; D012333:RNA, Messenger; D017472:Receptors, Interleukin-1; D014409:Tumor Necrosis Factor-alpha; D015847:Interleukin-4; D007371:Interferon-gamma; D016178:Granulocyte-Macrophage Colony-Stimulating Factor