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Quantification of Extracellular Double-stranded RNA Uptake and Subcellular Localization Using Flow Cytometry and
Tan A Nguyen1,2, Lachlan Whitehead1,2, Ken C Pang1,3,4,5
1The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.
Bio-Protocol
|July 21, 2021
Summary
This study presents a new method to track extracellular double-stranded RNA (dsRNA) uptake by cells. This technique visualizes dsRNA localization, aiding the study of innate immune responses to viral infection.
Area of Science:
- Immunology
- Virology
- Cell Biology
Background:
- Double-stranded RNA (dsRNA) is a key indicator of viral infection and a potent immune activator.
- Viral dsRNA can be released extracellularly and taken up by neighboring cells, triggering innate immune responses.
- Pattern recognition receptors (PRRs), like TLR3 and RLRs, detect dsRNA, initiating type I interferon production.
Purpose of the Study:
- To develop and validate a quantitative method for measuring extracellular dsRNA uptake.
- To visualize the subcellular localization of internalized dsRNA within cells.
- To provide insights into innate immune activation pathways triggered by viral dsRNA.
Main Methods:
- Quantitative measurement of extracellular dsRNA uptake using flow cytometry.
- Visualization of subcellular dsRNA localization using confocal microscopy.
- Utilizing dsRNA as a model PAMP to study cellular uptake mechanisms.
Main Results:
- Successfully quantified the uptake of extracellular dsRNA by mammalian cells.
- Visualized the intracellular trafficking and localization of internalized dsRNA.
- Demonstrated the utility of the developed methods in studying dsRNA-mediated immune signaling.
Conclusions:
- The described methods enable precise measurement of dsRNA uptake and localization.
- Understanding dsRNA internalization is crucial for deciphering innate immune activation during viral infections.
- This approach facilitates research into host-pathogen interactions and antiviral immunity.
