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Updated: Aug 25, 2026
Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation
Published on: December 15, 2017
Tumor necrosis factor-alpha analyzed within individual macrophages by combined immunocytochemistry and computer-aided
1Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Insights
This study developed a method combining immunocytochemistry and image analysis to measure intracellular tumor necrosis factor-alpha (TNF) in macrophages. Optimal conditions were found for activating and quantifying TNF production, highlighting early measurement importance.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Macrophages are key immune cells involved in inflammation and host defense.
- Tumor necrosis factor-alpha (TNF) is a critical cytokine with diverse roles in immunity and disease.
- Quantifying intracellular TNF production in individual macrophages is essential for understanding immune responses.
Purpose of the Study:
- To develop and optimize a method for quantifying intracellular tumor necrosis factor-alpha (TNF) production in macrophages.
- To determine the optimal conditions for macrophage activation and TNF production.
- To differentiate between intracellular, membrane-bound, and receptor-bound TNF.
Main Methods:
- Combining immunocytochemical staining with computer-aided image analysis.
- Optimizing protocols for cell activation (lipopolysaccharide + interferon-gamma), fixation, and image analysis.
- Utilizing acid stripping to distinguish different forms of TNF.
Main Results:
- Established optimal conditions for measuring intracellular TNF in macrophages.
- Demonstrated that thioglycolate-elicited peritoneal macrophages and alveolar macrophages readily produce TNF upon activation.
- Showed that both lipopolysaccharide (LPS) and interferon-gamma are necessary for increased intracellular TNF.
- Identified that maximum intracellular TNF levels occur 2-3 hours post-activation.
- Highlighted the importance of early measurement of different TNF forms.
Conclusions:
- The developed method allows for accurate quantitation of intracellular TNF in individual macrophages.
- Macrophage activation and TNF production vary depending on macrophage source and activation stimuli.
- Early assessment of TNF forms is crucial for a comprehensive understanding of its role in biological processes.
Abstract:
Immunocytochemical staining procedures were combined with computer-aided image analysis to quantitate the relative intracellular production of tumor necrosis factor-alpha (TNF) within individual macrophages. Optimal conditions for time and methods for the activation of TNF production, fixation of cells for optimal immunocytochemical staining, and image analysis methods were determined. Thioglycolate elicited peritoneal macrophages were readily activated to significantly increased levels of intracellular TNF, as early as 1 hr after activation with lypopolysaccharide (LPS) + interferon-gamma: maximum intracellular TNF was evident after 2-3 hr. Both LPS and interferon-gamma was necessary to increase intracellular TNF. Normal alveolar macrophages also readily produced increased intracellular TNF, but normal peritoneal and splenic macrophages were poorly activated to TNF production. Acid stripping of receptor bound TNF allowed discrimination between intracellular TNF/integral membrane TNF, and TNF-receptor-bound TNF. Results stress the importance of studying these TNF forms early after activation. Applications for TNF quantitation by these means are discussed.
