Influenza virus plaque assay

Collect and count MDCK cells

For the following steps, MDCK cells are maintained in 10 cm dishes in 10 ml volume of D10

Wash & collect cells

• Aspirate D10, add 4 ml sterile DPBS, aspirate

• Trypsinize cells with 1 ml 0.25% trypsin for 5-15 min at 37 C, tap gently to lift off

• Resuspend in 10 ml D10

• Count cells by adding 10 ul to counting slide and measure on TC20 or hemacytometer

Prepare enough cells to seed 12 well plates with 1 ml per well

• Resuspend cells to 3E5 cells/ml

• Add 1 ml per well in a 12 well plate

  Always fill all wells in a plate with liquid, even if all wells are not needed. This prevents evaporation effects

• Incubate cells at 37C, 5% CO₂ overnight

Prepare virus dilutions

Enveloped viruses are sticky and can adhere to plastic pipet tips. It's crucial to change tips between each dilution step, and pipet up & down a consistent number of times. For example, pipet up/down once, move liquid to next dilution, pipet up/down 8 times; discard tips, repeat.

From mouse lungs

Lungs should be collected in individual Eppendorf tubes and flash frozen. To flash freeze, submerge closed tube in liquid nitrogen. Frozen samples are stored at -80 C.

On day of infection, prepare lung homogenates as follows:

• Thaw lungs on ice for >1 h

• Weigh lungs, move to bead buster 2 ml eppie tubes that each contain 1 magnetic ball bearing

• Add 0.5 ml VGM to rinse original lung tube and add to 2 ml tube. Up to 1 ml liquid volume can be used

• Lyse lungs using Qiagen Tissue Lyser II

• Program 4: 3 cycles, 30 Hz, 30 s. In between cycles, move tubes to ice for ~1 minute

• Spin homogenates 10,000 x g at 4 C for 10 min, transfer supernatant (containing virus) to new eppie tube, continue to dilutions in step 2.2 below

• Transfer beads to 70% ethanol to sterilize, rinse thoroughly with DI H₂O, and store for later autoclaving

From tissue culture-grown virus stock

Infection inoculums for 12-well plates use 0.1 ml virus, when preparing dilutions, be sure to have adequate volume. Prepare 10-fold serial dilution series either in 96-well V-bottom plates or eppendorf tubes.

It may feel like less work to dilute in eppendorf tubes (less dilutions!) but by using a plate, you can use a multichannel pipette to simultaneously dilute samples. Plus the "technical replicate" portion is more faithful. E.g. if I'm just quick tittering 1-2 viruses I'll opt for eppendorf tubes. Most other situations I use 96-well plates.

Prepare in 96-well V-bottom plates (use each column as a technical singlet, ie, use 3 columns for triplicate):

    Add 135 ul VGM to all wells

    Add 15 ul stock solution to row A

    Dilute stock solution serially, transferring 15 ul stepwise

Prepare in eppendorf tubes (pull from eppendorf tubes to inoculate technical replicates):

    Add 360 ul VGM to eppendorf tubes

    Serially dilute 40 ul

Infect cells 1h 0m 0s

Inoculate

• Wash MDCK cells by aspirating, adding ~0.5 ml VGM per well

• Aspirate, add 0.1 ml virus inoculum

• Incubate at 37 C

  Critical to label plate with accurate description of dilution series/virus used

Infect for 1 h at 37C

• Rock plate every ~15 min by drastically tipping forward-back and side-side. Don't allow inoculum only to sit on the well edges or monolayer to dry out

Prepare overlay

Each well will receive 1 ml overlay, determine final volume needed

• Add 2X DMEM into 50 ml falcon tubes, add TPCK-trypsin to ~1 ug/ml (volume calculated after 2X diluted with Avicel). However, you need to first mix TPCK-trypsin with 2X DMEM before adding avicel, or the mixture won't be uniform concentration

• Dilute 2X DMEM 1:1 with Avicel

• Store these 50 ml tubes in 37 C water bath until ready to stop infection

Stop infection by adding overlay

• Add 1 ml overlay per well (no need to aspirate virus inoculum!)

  Critical to vigorously swirl plate after inoculum is added. The goal is to get an even mixture of inoculum/overlay. If you don't do this and maintain a liquid interface at the cell surface, plaques will be amorphous and hard to differentiate.

Incubate at 37 C for 3 days

72h 0m 0s

Some fast growing viruses like PR8 or WSN can be developed after 2 days, but 3 days is best to be safe.

Stop infection and visualize plaques with Crystal Violet

Aspirate wells

• Avicel-containing overlay can be aspirated into the trap (hooray!). In BSC, tip plate towards you and aspirate overlay, trying to collect it all.

Wash

• Add ~0.5 ml PBS (does not have to be sterile) to each well. I just lightly dispense across the plate without measuring

• Swirl the liquid in the wells to dislodge remaining overlay

• Aspirate

Fix

• Add ~0.5 ml 70% ethanol to each well

• Incubate for 10 minutes at room temperature

• Aspirate

0h 10m 0s

Stain

• Add ~0.5 ml Crystal Violet to each well, making sure that liquid completely coats the surface

• Incubate for 10 minutes at room temperature

• Aspirate into Crystal Violet trap in the Kajon lab fume hood

0h 10m 0s

Wash

• Add tap water to fill the wells of the plate and dump down the sink

• Repeat for a total of 4 washes

Dry

• Dry plates by leaving upside down at an angle to encourage air flow

• On top of bench pad in the fume hood

Titers are represented in plaque forming units (pfu) per ml stock

From animal infections, often represented as per g tissue. Be sure to weigh tissue so that you can back calculate later

Aim to count 10-30 plaques per well

Calculate titer by multiplying average number of plaques by reciprocal dilution factor and by inoculum volume

For example, and average of 3 plaques are observed in the 10^-5 dilution well

3 X 10⁵ [10^-5 dilution] X 10 [0.1 ml inoculum]

Save yourself some time by developing a spreadsheet template like this one