Immunofluorescence assay for the quantitative and qualitative evaluation of intracellular interleukin-8 in microtiter plates.
In order to monitor the effects of drugs on interleukin-8 (IL-8) production by cells, a microtiter plate assay that determines four parameters simultaneously was established (i) levels of secreted IL-8 (supernatant ELISA), (ii) levels of intracellular IL-8 (cell ELISA), (iii) intracellular localization (fluorescence microscopy), and (iv) the amount of cellular protein (colorimetric assay). The quantitative and qualitative determination of intracellular IL-8 was achieved by immunofluorescence using the ELF-detection system (Molecular Probes, Eugene, OR), which is approximately 10 times more sensitive than conventional immunofluorescence detection systems. Thus, a 32x objective magnification (without immersion oil) is sufficient to precisely assess the subcellular localization of IL-8. Experiments were carried out with human umbilical vein endothelial cells. Drugs interfering with transcription or translation and inhibitors of protein kinase C inhibited both production and secretion of IL-8. Brefeldin A (BFA), colchicine, and the HMGCoA-reductase inhibitor fluvastatin disrupted the characteristic Golgi localization of IL-8, but only BFA inhibited its secretion. This assay can therefore be used to distinguish drugs that inhibit both IL-8 synthesis and secretion from those that inhibit IL-8 secretion only. It can easily be adapted to other cellular proteins for which a sensitive detection method is required.
Wolff
B
B
Sandoz Research Institute, Vienna, Austria.
Zsak
M
M
Rabeck
C
C
D020126:Brefeldin A; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology; D003517:Cyclopentanes / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005229:Fatty Acids, Monounsaturated / Q000494:pharmacology; D005455:Fluorescent Antibody Technique / Q000379:methods*; D000077340:Fluvastatin; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007211:Indoles / Q000494:pharmacology; D016209:Interleukin-8 / Q000032:analysis*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology
D003517:Cyclopentanes; D004791:Enzyme Inhibitors; D005229:Fatty Acids, Monounsaturated; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007211:Indoles; D016209:Interleukin-8; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A; D000077340:Fluvastatin; D003078:Colchicine