Lung Homogenization
Liyen Loh, Marios Koutsakos, Katherine Kedzierska, Timothy S C Hinks, Bonnie van Wilgenburg, Huimeng Wang, Alexandra J. Corbett, Zhenjun Chen
Abstract
This is part 3.4 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
Collect the lungs into 2mL.
For homogenization, place the lung and the 2mL into 10 mL falcon tubes with lids ( see Note 16 ).
Prepare 10 or 15 mL Falcon tubes with 2 × 5mL for cleaning the homogenization probe initially and 1 tube containing HBSS. For each group of samples, prepare further 1 × 5mL and 1 × 5mL, and for the final probe clean set up 2 × 5mL.
Homogenize the sample using a homogenizer, mounted on a retort stand with the probe set to medium for 0h 0m 30s per sample. Keep samples On ice ( see Note 17 ).
Centrifuge the samples at 1000x g.
Using a 1 mL pipette, carefully draw up approximately 1mL(a little bit more is good), avoiding the pellet and fatty residue on top. Divide this volume into two 1.5 mL Eppendorf tubes. Store at -80°C for subsequent plaque assays.
