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Journal of Histochemistry and Cytochemistry, Vol. 45, 935-946, Copyright © 1997 by The Histochemical Society, Inc.

ARTICLE

Detection of Intracellular Interleukin-8 in Human Mast Cells: Flow Cytometry as a Guide for Immunoelectron Microscopy

Andreas Grützkaua, Sabine Krüger-Krasagakesa, Heidi Kögela, Annelie Möllera, Undine Lipperta, and Beate M. Henza
a Department of Dermatology, Virchow Clinics, Humboldt University of Berlin, Berlin, Germany

Correspondence to: Andreas Grützkau, Virchow Klinikum Dermatologie, Augustenburger Platz 1, 13353 Berlin, Germany.

The chemokine interleukin-8 (IL-8) mediates infiltration and adhesion of neutrophils during inflammatory processes. We have previously shown that this cytokine can be produced and released by normal and leukemic human mast cells (HMC-1 cells). To assess whether and to what extent this cytokine is stored intracellularly, we investigated production and localization of IL-8 at the single-cell level by combined use of flow cytometry (FACS) and immunoelectron microscopy. Conditions necessary for optimal fixation and permeabilization of HMC-1 cells were determined by measuring changes in cell-specific light scatter parameters and by estimating cellular uptake of propidiumiodide (PI). In this way, we were able to detect IL-8 with a monoclonal antibody in stimulated cells that were microwave-fixed with a combination of paraformaldehyde (4%) and glutaraldehyde (0.1%), followed by permeabilization with saponin (0.025%). FACS analysis revealed time-dependent synthesis of IL-8 with at most 50% positively stained cells at 8-12 hr after stimulation. For pre-embedding immunogold electron microscopy, cells were treated according to the protocol established by flow cytometry. IL-8 was found to be located in specific cytoplasmic, electron-dense granules of stimulated HMC-1 cells. These results confirm and extend our previous findings by demonstrating IL-8 expression in HMC-1 cells at the single-cell level. In addition, we propose that quantitative FACS can be reliably used in a timesaving manner to establish appropriate conditions for pre-embedding immunoelectron microscopy of intracellular antigens. (J Histochem Cytochem 45:935-945, 1997)

Key Words: mast cells, HMC-1 cells, interleukin-8, immunoelectron microscopy, pre-embedding immunocytochemistry, flow cytometry, colloidal gold


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