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Protocol for induction and characterization of microglia extracellular traps in murine and human microglia cells
Ishan Agrawal1, Nidhi Sharma1,2, Shivanjali Saxena1
1Department of Bioscience and Bioengineering, Indian Institute of Technology Jodhpur, Jodhpur, Rajasthan 342037, India.
Abstract:
Extracellular traps (ETs) are composed of decondensed chromatin and are embedded with various antimicrobial proteins like myeloperoxidase and histones. Recently, we reported that dopamine (DA) induces ETs in BV2 microglia cell line and primary adult human microglia in a manner independent of cell death, reactive oxygen species, and actin polymerization. This protocol details how to characterize DA-induced ETs in BV2 microglia and human microglia. The protocols for characterization of ETs may also be used for other adherent cell lines. For complete details on the use and execution of this protocol, please refer to Agrawal et al. (2021).
Insights
Dopamine (DA) triggers the release of extracellular traps (ETs) from microglia cells. This protocol outlines methods to identify these DA-induced ETs in both cell lines and primary human cells.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Extracellular traps (ETs) are crucial components of the innate immune system, characterized by decondensed chromatin decorated with antimicrobial proteins.
- Microglia, the resident immune cells of the central nervous system, play a vital role in neuroinflammation and host defense.
- Previous research has indicated that dopamine (DA) can influence microglial functions.
Purpose of the Study:
- To provide a detailed protocol for characterizing dopamine (DA)-induced extracellular traps (ETs) in microglial cells.
- To establish methods for identifying ETs in the BV2 microglia cell line and primary adult human microglia.
- To offer a versatile protocol applicable to other adherent cell lines for ET characterization.
Main Methods:
- Utilizing BV2 microglia cell line and primary adult human microglia.
- Inducing extracellular trap formation using dopamine (DA).
- Characterizing DA-induced ETs through established microscopy and biochemical techniques, ensuring independence from cell death, reactive oxygen species, and actin polymerization.
Main Results:
- Dopamine (DA) was confirmed to induce the formation of extracellular traps (ETs) in both BV2 microglia and primary human microglia.
- The induction of ETs by DA was demonstrated to be independent of cell death, reactive oxygen species (ROS) production, and actin polymerization.
- The study provides a reliable protocol for the visualization and analysis of these specific DA-induced microglial ETs.
Conclusions:
- Dopamine (DA) is a novel inducer of extracellular traps (ETs) in microglia, operating through a unique mechanism.
- The developed protocol enables robust characterization of DA-induced ETs, offering a valuable tool for neuroimmunology research.
- These findings contribute to understanding the complex interplay between neurotransmitters and innate immune responses in the brain.
