Viral Plaque Assay
Liyen Loh, Marios Koutsakos, Katherine Kedzierska, Timothy S C Hinks, Bonnie van Wilgenburg, Huimeng Wang, Alexandra J. Corbett, Zhenjun Chen
Abstract
This is part 3.6 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.
Abstract: Viral plaque assays are used to determine influenza viral titers. A diluted solution of egg-adapted Influenza A viruses/lung-infected tissue homogenates are applied to a six-well tissue culture dish containing a monolayer of Madin-Darby canine kidney (MDCK) cells. The infected MDCK cells grow under a semisolid overlay medium (agar) containing trypsin. A plaque is produced when a virus particle infects a cell, replicates, and then kills the cell. This process can be repeated several times as surrounding cells can be infected by newly replicated virus and killed. When visualized by eye, plaques appear as white spots. The assay is measured in PFU/mL.
Attachments
Steps
Passaging MDCK cells:
Warm up MDCK cell media, trypsin–versene, and PBS at 37°C.
Check the confluency of MDCK cells, aspirate the medium, add 10mL, aspirate the medium, and repeat wash.
Discard PBS, add 2–3 mL of trypsin–versene (stored -20°C) to MDCK monolayers, and incubate at 37°C for 0h 5m 0s. After 0h 5m 0s tap the flasks, and incubate for longer if required (maximum 0h 15m 0s).
In the meantime, add 15mL to fresh T75 flasks.
Add MDCK cell media to a total volume of 10mL to the trypsinized cells, and transfer cells to a 10-mL tube.
Count cells using a hemocytometer.
Set up multiple T75 flasks with different cell densities to determine the growth pattern of MDCK cells. Generally ~3–5 × 105for 3-day split.
Incubate at 37°C, 5%.
Amplification of MDCK cells for plaque assay:
Warm up MDCK cell media, trypsin–versene, and PBS at 37°C.
Check the confluency of MDCK cells, aspirate the medium, add 10mL, aspirate the medium, and repeat wash.
Discard PBS, add 2mL–3mL (stored -20°C) to MDCK monolayers, and incubate at 37°C for 0h 5m 0s. After 0h 5m 0s tap the flasks, and incubate for longer if required (maximum 0h 15m 0s).
In the meantime, add 40mL to fresh T175 flasks. Set up one T175 flask of MDCK cells per ~4 plates for plaque assay. Each 6-well plate assays 3 viral dilutions (as dilutions are done in duplicate).
Add MDCK cell media to a total volume of 10mL to trypsinized cells, and transfer cells to a 10-mL tube.
Count cells using a hemocytometer.
Add ~7–8 × 105 cells per T175 flask for 3-day culture.
Seeding flat-bottomed 6-well tissue culture (TC6) plates for plaque assay:
Warm up MDCK cell media, trypsin–versene, and PBS at 37°C.
Check the confluency of MDCK cells, aspirate the medium, add 10mL, aspirate the medium, and repeat the wash.
Discard PBS, add 5mL–8mL (-20°C) to MDCK monolayers in T175 flask, incubate at 37°C for 0h 5m 0s. After 0h 5m 0s, tap bottles, incubate for longer if required (maximum 0h 15m 0s).
Add 17mL–20mL to each flask (total 25mL including 5mL–8mL) to inhibit trypsin–versene.
Pool cells into one flask.
Count the cells, adjust the concentration to 3.3 × 105 cells/mL 5 cells/mL.
Add 3mL of 3.3 × 105cells/mL to each well of TC6 plates (~1 × 106/well), swirl plates gently to distribute cells evenly. Include a negative control plate.
Incubate cells at 37°C, 5% 0h 15m 0s. Aim for monolayers to be confluent in 6-well plates for assay.
Plaque assay
Warm up SF-DMEM at 37°C.
Prepare dilutions of samples to be titrated. This can be done in a 96-well flat-bottom plate.
Cells will be infected with 150µL of each dilution in duplicate, so a minimum of 300µL of each dilution is required. A 96-well flat-bottom plate can hold ~350 μL/well . 35µL would be added to 315µL for a final volume of 350µL (35 in 350 = 1:10 dilution). If a sample is to be plaqued neat, add 350µL to the first well.
Add 315µL in each well of 96-well plate. A multichannel can be used and add 157µL and 158µL (total 315µL) to each well.
Add 35µL to the first well with media (tenfold dilution) and continue serial tenfold dilutions by transferring 35µL across wells, changing tips between dilutions.
For titration of viral stocks use dilutions from 10-4 to 10-6. 10-1 can be used as a positive control. Half-log dilutions can also be performed. For titration of mouse lung homogenates, generally:
(a) Days 1–5: 10-1 to 10-3.
(b) Days 6–10: neat to 10-2 ( see Note 26 ).
Wash MDCK cells with 1mL–2mL of PBS/well.
Infect cells with 150 μL/well of the appropriate dilution, swirl gently to cover all cells and incubate at 37°C, 5% for 1h 0m 0s, shake gently every 15 min .
In the meantime, prepare overlay media, best to start doing so in the beginning of the 1 h incubation of MDCK cells.
Weigh out 1.8g into 200 mL glass bottle, add 100mL (1.8%), and melt in microwave. Store in a 55°C water bath.
Aliquot 50mL into 50 mL tubes (need 2 × 50 mL aliquots of 2 × L-15 medium and 1 bottle (100mL) of 1.8%) and store in 37°C water bath.
Thaw trypsin–versene at 40°C. Thaw a 200µL aliquot/50 mL of 2 × L-15.
After 1 h incubation of MDCK cells with sample: Add 200µL to each 50 mL tube of 2 × L-15.
Make overlay media by adding 100mL to 100mL and mix well ( see Note 27 ).
Add 3mL/well and leave at Room temperature until it sets.
Incubate upside-down at 37°C, 5% for 3 days. Plaques maybe visible by the end of day 2 and the plates can be incubated till day 4 if plaques are too small on day 3.
Count the plaques. This can be done by holding the plates against the light. Alternatively, remove agarose overlay and stain with crystal violet. To stain, cover the cells with a minimal amount of crystal violet solution for ~0h 15m 0s. Rock plates if necessary to ensure even coverage. Gently wash off the crystal violet stain with water. Once fixed, stained, and dried, store plaques indefinitely for future analysis.
Calculate viral titer in PFU/mL: Average count of duplicate well × Dilution factor × (1000/150) = PFU/mL (multiply lung homogenate counts by 2 to give total viral load, as lungs were taken and homogenized in 2mL).
