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Published on: July 23, 2012
Assessing mRNA translation in mouse adult microglia and bone-marrow-derived macrophages
Ignazio Antignano1, Lily Keane1, Melania Capasso1
1Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), 53127 Bonn, Germany.
Abstract:
Protein synthesis, or mRNA translation, is the biological process through which genetic information stored in messenger RNAs is encoded into proteins. Here, we present an optimized protocol for assessing the translation rate in mouse adult microglia and cultured bone-marrow-derived macrophages. We describe steps for isolating cells, treating them with a puromycin-analog probe, and fluorescently labeling the puromycylated-polypeptide chains. We then detail their quantification by flow cytometry or with a fluorescent plate reader. For complete details on the use and execution of this protocol, please refer to Keane et al. (2021).1.
Insights
This study optimizes a method to measure protein synthesis rates in mouse immune cells. The protocol uses a puromycin probe to quantify translation in microglia and macrophages, aiding research into cellular function.
Area of Science:
- Molecular Biology
- Immunology
- Cell Biology
Background:
- Protein synthesis (mRNA translation) is crucial for cellular function, converting genetic code into proteins.
- Measuring translation rates in specific cell types, like immune cells, is vital for understanding their roles in health and disease.
Purpose of the Study:
- To present an optimized protocol for assessing mRNA translation rates.
- To provide a reliable method for quantifying protein synthesis in mouse adult microglia and bone-marrow-derived macrophages.
Main Methods:
- Isolation of primary mouse microglia and cultured macrophages.
- Treatment of cells with a puromycin-analog probe to label newly synthesized proteins.
- Fluorescent labeling of puromycylated polypeptide chains.
- Quantification using flow cytometry or a fluorescent plate reader.
Main Results:
- An optimized and detailed protocol for measuring translation rates in specific immune cell populations.
- Demonstration of puromycin incorporation and subsequent fluorescent detection.
- Establishment of quantitative measurement techniques for protein synthesis.
Conclusions:
- The presented protocol offers a robust method for assessing protein synthesis in mouse microglia and macrophages.
- This technique facilitates the study of translation dynamics in these critical immune cells.
- The protocol is adaptable for various research applications requiring measurement of cellular translation activity.
