Influenza A Virus Infection
Liyen Loh, Marios Koutsakos, Katherine Kedzierska, Timothy S C Hinks, Bonnie van Wilgenburg, Huimeng Wang, Alexandra J. Corbett, Zhenjun Chen
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
Steps
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%.
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:
10µL+990µL. - Dilution 2: 1/100:
10µL+990µL. - Dilution 3: 1/100:
10µL+990µL. - Dilution 4: ½: (depends on volume required, e.g., 20 inoculations = 1000 μL)
500µLand500µL.
Mix virus with vortex before administering the inoculum.
Infect mice i.n. with 50µL under isoflurane anesthesia ( see Note 7 ).
Allow mice to recover and monitor mice for until recovery (typically 10 days) ( see Note 15 ).
