Format

Send to

Choose Destination
See comment in PubMed Commons below
Biotech Histochem. 2000 May;75(3):101-9.

Identification of rat IL-1beta, IL-2, IFN-gamma and TNF-alpha in activated splenocytes by intracellular immunostaining.

Author information

1
Centre of Molecular Medicine, Karolinska Institute, and Department of Pediatric Rheumatology, Astrid Lindgren's Children's Hospital, Karolinska Hospital, Stockholm, Sweden. karin.palmblad@cmm.ki.se

Abstract

We have developed a sensitive three-step indirect immunofluorescence method to identify individual rat cells that produce cytokines including IL-1beta, IL-2, IFN-gamma and TNF-alpha. Cultured rat splenocytes were polyclonally activated to cytokine synthesis by mitogens such as lipopolysaccharide or a combination of a protein kinase C activator (phorbol 12-myristate 13-acetate) and a calcium ionophore (ionomycin). Careful selection of either antigen affinity-purified polyclonal or monoclonal cytokine-detecting antibodies combined with gentle fixation and permeabilization of the cells enabled discrimination of cytokine-producing cells based on distinct morphological staining criteria. Cells making IL-2, IFN-gamma and TNF-alpha could be identified by a characteristic, intracellular, rounded, juxtanuclear immunofluorescence signal. This staining pattern reflected the accumulation of the intracellularly processed cytokines in the Golgi organelle of producer cells. The staining of cells that synthesized IL-1beta, which is not transported intracellularly via the endoplasmatic reticulum-Golgi pathway, generated a different, but distinct and reproducible staining pattern, IL-1beta producing macrophages expressed intense nuclear immunofluorescence with additional reticular, cytoplasmic signals. Furthermore, the use of biologically neutralizing detecting antibodies against the cytokines under study prevented recognition of surface-stained target cells that had bound secreted cytokines by cytokine-specific receptors. This modified staining technology enabled analysis of the kinetic pattern and the frequency of cytokine-producing cells in cultures of rat splenocytes after various modes of polyclonal activation.

PMID:
10950171
[Indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Loading ...
    Support Center