TITLE: Viral Plaque Assay
AUTHORS: Timothy S C Hinks, Bonnie van Wilgenburg, Huimeng Wang, Liyen Loh, Marios Koutsakos, Katherine Kedzierska, Alexandra J. Corbett, Zhenjun Chen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is part 3.6 of the "Study of MAIT Cell Activation in Viral Infections In Vivo" collection of protocols.</div><br/><div class = "text-block"><span style = "font-weight:bold;">Collection Abstract: </span><span>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.</span></div><br/><div class = "text-block"><span style = "font-weight:bold;">Abstract: </span><span>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.</span></div></div>

[STEPS]
?. [Passaging MDCK cells:]
Warm up MDCK cell media, trypsin–versene, and PBS at .
37 °C
?. [Passaging MDCK cells:]
Check the confluency of MDCK cells, aspirate the medium, add , aspirate the medium, and repeat wash.
[PBS]
?. [Passaging MDCK cells:]
Discard PBS, add 2–3 mL of trypsin–versene (stored ) to MDCK monolayers, and incubate at  for . After  tap the flasks, and incubate for longer if required (maximum ).
-20 °C
37 °C
?. [Passaging MDCK cells:]
In the meantime, add  to fresh T75 flasks.
[MDCK cell media]
?. [Passaging MDCK cells:]
Add MDCK cell media to a total volume of  to the trypsinized cells, and transfer cells to a 10-mL tube.
10 mL
?. [Passaging MDCK cells:]
Count cells using a hemocytometer.
?. [Passaging MDCK cells:]
Set up multiple T75 flasks with different cell densities to determine the growth pattern of MDCK cells. Generally ~3–5 × 105 for 3-day split.
?. [Passaging MDCK cells:]
Incubate at , .
37 °C
[CO2]
?. [Amplification of MDCK cells for plaque assay:]
Warm up MDCK cell media, trypsin–versene, and PBS at .
37 °C
?. [Amplification of MDCK cells for plaque assay:]
Check the confluency of MDCK cells, aspirate the medium, add , aspirate the medium, and repeat wash.
[PBS]
?. [Amplification of MDCK cells for plaque assay:]
Discard PBS, add – (stored ) to MDCK monolayers, and incubate at  for . After  tap the flasks, and incubate for longer if required (maximum ).
2 mL
[trypsin–versene]
-20 °C
37 °C
?. [Amplification of MDCK cells for plaque assay:]
In the meantime, add  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).
[MDCK cell media]
?. [Amplification of MDCK cells for plaque assay:]
Add MDCK cell media to a total volume of  to trypsinized cells, and transfer cells to a 10-mL tube.
10 mL
?. [Amplification of MDCK cells for plaque assay:]
Count cells using a hemocytometer.
?. [Amplification of MDCK cells for plaque assay:]
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
?. [Seeding flat-bottomed 6-well tissue culture (TC6) plates for plaque assay:]
Check the confluency of MDCK cells, aspirate the medium, add , aspirate the medium, and repeat the wash.
[PBS]
?. [Seeding flat-bottomed 6-well tissue culture (TC6) plates for plaque assay:]
Discard PBS, add – () to MDCK monolayers in T175 flask, incubate at  for . After , tap bottles, incubate for longer if required (maximum ).
5 mL
[ trypsin–versene]
-20 °C
37 °C
?. [Seeding flat-bottomed 6-well tissue culture (TC6) plates for plaque assay:]
Add – to each flask (total  including –) to inhibit trypsin–versene.
17 mL
[MDCK cell media]
25 mL
5 mL
[trypsin]
?. [Seeding flat-bottomed 6-well tissue culture (TC6) plates for plaque assay:]
Pool cells into one flask.
?. [Seeding flat-bottomed 6-well tissue culture (TC6) plates for plaque assay:]
Count the cells, adjust the concentration to 3.3 × 105 cells/mL.
?. [Seeding flat-bottomed 6-well tissue culture (TC6) plates for plaque assay:]
Add  of 3.3 × 105 cells/mL to each well of TC6 plates (~1 × 106/well), swirl plates gently to distribute cells evenly. Include a negative control plate.
3 mL
?. [Seeding flat-bottomed 6-well tissue culture (TC6) plates for plaque assay:]
Incubate cells at , . Aim for monolayers to be confluent in 6-well plates for assay.
37 °C
[CO2]
?. [Plaque assay]
Warm up SF-DMEM at .
37 °C
?. [Plaque assay]
Prepare dilutions of samples to be titrated. This can be done in a 96-well flat-bottom plate.
?. [Plaque assay]
Cells will be infected with  of each dilution in duplicate, so a minimum of  of each dilution is required. A 96-well flat-bottom plate can hold ~350 μL/well.  would be added to  for a final volume of  (35 in 350 = 1:10 dilution). If a sample is to be plaqued neat, add  to the first well.
150 µl
300 µl
[of samples]
[media]
350 µl
[of sample]
?. [Plaque assay]
Add  in each well of 96-well plate. A multichannel can be used and add  and  (total ) to each well.
[SF-DMEM]
157 µl
158 µl
315 µl
?. [Plaque assay]
Add  to the first well with media (tenfold dilution) and continue serial tenfold dilutions by transferring  across wells, changing tips between dilutions.
[sample]
35 µl
?. [Plaque assay]
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).
?. [Plaque assay]
Wash MDCK cells with – of PBS/well.
1 mL
2 mL
?. [Plaque assay]
Infect cells with 150 μL/well of the appropriate dilution, swirl gently to cover all cells and incubate at ,  for , shake gently every 15 min.
37 °C
[CO2]
?. [Plaque assay]
In the meantime, prepare overlay media, best to start doing so in the beginning of the 1 h incubation of MDCK cells.
?. [Plaque assay]
Weigh out  into 200 mL glass bottle, add  (), and melt in microwave. Store in a  water bath.
[agarose]
[sterile water]
[agarose]
55 °C
?. [Plaque assay]
Aliquot  into 50 mL tubes (need 2 × 50 mL aliquots of 2 × L-15 medium and 1 bottle () of ) and store in  water bath.
[2 × L-15 medium]
100 mL
[agarose]
37 °C
?. [Plaque assay]
Thaw trypsin–versene at . Thaw a  aliquot/50 mL of 2 × L-15.
40 °C
200 µl
?. [Plaque assay]
After 1 h incubation of MDCK cells with sample: Add  to each 50 mL tube of 2 × L-15.
[trypsin]
?. [Plaque assay]
Make overlay media by adding  to  and mix well (see Note 27).
[2 × L-15 + trypsinsolution]
[1.8% agarose]
?. [Plaque assay]
Add /well and leave at  until it sets.
[overlay medium]
0 Room temperature
?. [Plaque assay]
Incubate upside-down at ,  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.
37 °C
[CO2]
?. [Plaque assay]
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 ~. 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.
?. [Plaque assay]
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 ).
[RPMI]