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Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes
Published on: December 20, 2014
Immunofluorescence assay for the quantitative and qualitative evaluation of intracellular interleukin-8 in microtiter
Insights
A novel microtiter plate assay simultaneously monitors interleukin-8 (IL-8) production, secretion, localization, and protein levels. This method distinguishes drugs inhibiting IL-8 synthesis from those affecting secretion only.
Area of Science:
- Cell biology
- Molecular biology
- Drug discovery
Background:
- Interleukin-8 (IL-8) is a key cytokine in inflammatory responses.
- Monitoring IL-8 production and secretion is crucial for understanding cellular responses to drugs.
- Existing methods may lack the sensitivity or multiplexing capability for comprehensive analysis.
Purpose of the Study:
- To establish a versatile microtiter plate assay for simultaneous monitoring of IL-8.
- To quantify secreted and intracellular IL-8 levels.
- To determine IL-8's subcellular localization and total cellular protein content.
Main Methods:
- Developed a multiplexed microtiter plate assay.
- Utilized supernatant ELISA for secreted IL-8.
- Employed cell ELISA and ELF-detection immunofluorescence for intracellular IL-8.
- Incorporated fluorescence microscopy for subcellular localization and colorimetric assay for protein content.
Main Results:
- The assay successfully measured four parameters: secreted IL-8, intracellular IL-8, localization, and protein levels.
- ELF-detection provided high sensitivity for intracellular IL-8 determination.
- Drugs affecting transcription, translation, or protein kinase C inhibited IL-8 production and secretion.
- Brefeldin A, colchicine, and fluvastatin disrupted Golgi localization, with BFA uniquely inhibiting secretion.
Conclusions:
- The developed assay enables simultaneous, sensitive, and quantitative assessment of IL-8.
- It effectively differentiates drugs based on their impact on IL-8 synthesis versus secretion.
- The assay is adaptable for studying other cellular proteins requiring sensitive detection.
Abstract:
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.
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