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A convenient and sensitive cytotoxicity assay for macrophage activating factor
Abstract:
A convenient procedure is described for assaying macrophage activating factor (MAF) in lymphokine preparations. This procedure utilizes thioglycollate-elicited peritoneal macrophages from C57BL/6J mice as effector cells and [3H]thymidine-labeled L929 target cells. A novel component of the cytotoxicity assay is the inclusion of a 20 min terminal incubation of the cultures with 10 micrograms/ml pancreatic deoxyribonuclease to facilitate the release of radioactivity from killed target cells. This method routinely gives a 10-20-fold greater release of [3H]thymidine from cultures containing MAF than from cultures containing macrophages and target cells alone. Additional advantages of this procedure are that it is relatively rapid, killing can be monitored microscopically, and MAF assays can be performed using macrophage concentrations as low as 1 X 10(5)/cm2, using effector to target cell ratios as low as 1:1, and using lymphokine dilutions of 10(-3) - 10(-4).
Insights
This study presents a new, rapid assay for macrophage activating factor (MAF) using [3H]thymidine-labeled cells and deoxyribonuclease. The method enhances MAF detection sensitivity by 10-20 fold.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Macrophage activating factor (MAF) plays a crucial role in immune responses.
- Accurate and sensitive assays are essential for studying MAF activity in lymphokine preparations.
- Existing methods may lack efficiency or require specific conditions.
Purpose of the Study:
- To develop a convenient and sensitive assay for quantifying macrophage activating factor (MAF) in lymphokine preparations.
- To optimize the detection of MAF-induced macrophage activation and cytotoxicity.
Main Methods:
- Utilized thioglycollate-elicited peritoneal macrophages from C57BL/6J mice as effector cells.
- Employed [3H]thymidine-labeled L929 cells as target cells.
- Incorporated a novel 20-minute terminal incubation with pancreatic deoxyribonuclease (10 µg/ml) to enhance radioactivity release from lysed target cells.
Main Results:
- The assay demonstrated a 10-20 fold greater release of [3H]thymidine in the presence of MAF compared to controls.
- The procedure is rapid and allows for microscopic monitoring of target cell killing.
- Effective MAF assays were achievable with low macrophage concentrations (1 x 10^5/cm2), low effector:target ratios (1:1), and high lymphokine dilutions (10^-3 - 10^-4).
Conclusions:
- The described assay provides a convenient, sensitive, and rapid method for quantifying MAF activity.
- This optimized procedure facilitates the study of MAF in various immunological research contexts.
- The enhanced sensitivity and efficiency make it suitable for a wide range of experimental conditions.