Sep 22, 2021
Influenza A Virus Infection
- Timothy S C Hinks1,
- Bonnie van Wilgenburg2,
- Huimeng Wang3,
- Liyen Loh3,
- Marios Koutsakos3,
- Katherine Kedzierska3,
- Alexandra J. Corbett3,
- Zhenjun Chen3
- 1Respiratory Medicine Unit, Nuffield Department of Medicine Experimental Medicine, University of Oxford, Oxfordshire, UK;
- 2Peter Medawar Building for Pathogen Research and Translational Gastroenterology Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK;
- 3Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville, Australia
- Springer Nature Books
Protocol Citation: Timothy S C Hinks, Bonnie van Wilgenburg, Huimeng Wang, Liyen Loh, Marios Koutsakos, Katherine Kedzierska, Alexandra J. Corbett, Zhenjun Chen 2021. Influenza A Virus Infection. protocols.io https://dx.doi.org/10.17504/protocols.io.bmgyk3xw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: September 17, 2020
Last Modified: September 22, 2021
Protocol Integer ID: 42232
Keywords: Virus, MAIT cell, Flow cytometry, MR1-tetramer, Infection, Mouse ,
Abstract
This is part 3.3 of the "Study of MAIT Cell Activation in Viral Infections In Vivo" collection of protocols.
Collection Abstract: MAIT cells are abundant, highly evolutionarily conserved innate-like lymphocytes expressing a semi-invariant T cell receptor (TCR), which recognizes microbially derived small intermediate molecules from the riboflavin biosynthetic pathway. However, in addition to their TCR-mediated functions they can also be activated in a TCR-independent manner via cytokines including IL-12, -15, -18, and type I interferon. Emerging data suggest that they are expanded and activated by a range of viral infections, and significantly that they can contribute to a protective anti-viral response. Here we describe methods used to investigate these anti-viral functions in vivo in murine models. To overcome the technical challenge that MAIT cells are rare in specific pathogen-free laboratory mice, we describe how pulmonary MAIT cells can be expanded using intranasal bacterial infection or a combination of synthetic MAIT cell antigen and TLR agonists. We also describe protocols for adoptive transfer of MAIT cells, methods for lung homogenization for plaque assays, and surface and intracellular cytokine staining to determine MAIT cell activation.
Attachments
Materials
For materials, please refer to the Guidelines section of the '"Study of MAIT Cell Activation in Viral Infections In Vivo" collection.
Safety warnings
Personal protective equipment (PPE) should be worn at all times (gloves, lab coat, & eye protection) (see Notes 3 and 4).
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Thaw virus vial rapidly in a 37 °C water bath until all ice crystals have melted.
Decontaminate the outer surface of the vial with 70 % ethanol .
Perform serial dilutions in sterile PBS to achieve the desired inoculum. For example:
(a) If titer of PR8 stock = 1.0 × 109 plaque-forming units (PFU)/mL, require 25 PFU/inoculum (see Note 14).
(b) Volume of inoculum required for intranasal infection = 50 μL/inoculum.
(c) 25 PFU/50 μL = 500 PFU/mL, dilution required from virus stock DF = 2,000,000.
- Dilution 1: 1/100: 10 µL virus stock + 990 µL PBS .
- Dilution 2: 1/100: 10 µL dilution 1 + 990 µL PBS .
- Dilution 3: 1/100: 10 µL dilution 2 + 990 µL PBS .
- Dilution 4: ½: (depends on volume required, e.g., 20 inoculations = 1000 μL) 500 µL dilution 3 and 500 µL PBS .
Mix virus with vortex before administering the inoculum.
Infect mice i.n. with 50 µL under isoflurane anesthesia (see Note 7).
Allow mice to recover and monitor mice for until recovery (typically 10 days) (see Note 15).