Detection of intracellular interleukin-8 in human mast cells: flow cytometry as a guide for immunoelectron microscopy.
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.
Grützkau
A
A
Department of Dermatology, Virchow Clinics, Humboldt University of Berlin, Germany.
Krüger-Krasagakes
S
S
Kögel
H
H
Möller
A
A
Lippert
U
U
Henz
B M
BM
D000911:Antibodies, Monoclonal; D002451:Cell Compartmentation; D002463:Cell Membrane Permeability; D003594:Cytoplasmic Granules / Q000737:chemistry / Q000648:ultrastructure; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006801:Humans; D016209:Interleukin-8 / Q000276:immunology / Q000302:isolation & purification*; D008407:Mast Cells / Q000737:chemistry* / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron / Q000379:methods*; D016707:Tissue Fixation
D000911:Antibodies, Monoclonal; D016209:Interleukin-8