GHV-68 Plaque Assay
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Plaque assay for gHV-68 using 3T12 fibroblasts.
- X μL reagent 2
- component A (reagent 2 is made up of multiple components)
- component B
- equipment 1
- equipment 2
- Trypsinize 3T12's (1 confluent T175 will yield approx. 10-40x106, cells) and resuspend at 2.5-2.5x106/ml in DMEM-10.
- Aliquot 2/ml/well (3-5x105 cells/well) into 6-well plates. Don't swirl the cells, as you may end up with all your 3T12's in the center of the well.
- Need one 6-well plate per viral sample. (This will let you test 6 logs of dilution)
- Prepare methylcellulose, if necessary.
- Freeze/thaw/freeze your viral sample, if necessary.
- Thaw your sample that you want to titer and warm methylcellulose to 37° C.
- Perform 10-fold serial dilutions in a round-bottom 96-well plate.
- Each column represents a different sample of virus to be diluted.
- Each row represents a 10-fold dilution of the virus, ie—row A is 100, row B is 10-1, row C is 10-2, etc.
- Don't forget to include a mock (media only) and a stock viral solution (previously titered) as controls.
- Aliquot 225 ul/well fresh media in row B-H.
- Aliquot 250 ul/well neat virus in row A.
- Transfer 25 ul with multichannel pipette from row A to row B.
- Pipette up and down 15 times to mix thoroughly the contents of the well.
- Change tips.
- Repeat for row B to row C, etc.
- Discard media from 6-well plates into a beaker in the hood.
- Transfer 200 ul from each of the wells in the dilution matrix into one well of a 6-well plate. Pipette the sample down the wall of the well of the 6-well plate; don't squirt it into the middle of the monolayer of cells, since this can make a false plaque. Start at the highest dilution (lowest concentration of virus) in the 96-well plate and move up a row to more and more concentrated virus using the same tip. One must change tips, obviously, when moving to a new virus sample (new column).
- Optional: Add an additional 200 ul/well of D-10 to each well for a total of 400 ul/well. This extra volume prevents drying of the monolayer during the adsorption step.
- Adsorb the virus at 37C for 1-2 hrs; use frequent rocking (every 10-15 min by hand) to avoid drying the cells. Note: Previous protocols have done the dilutions in eppendorf tubes and the adsorption step with 500 ul per well and rocked the plates on the automatic rocker at RT. I've had problems with the automatic rocker and the lower volume (400 ul/well) that is suggested in this protocol. I think the rocker may actually promote drying of the center of the well by drawing the fluid to the side of the well via capillary action.
- Overlay approx 5 ml warm methylcellulose per well.
- Hand rock each plate gently to mix the virus-containing adsorption media with the media in the methylcellulose. This is in an attempt to avoid pockets of virus forming between the layer of cells and the methylcellulose.
- Incubate 37C, 5% CO2 for 6 d.
- Add neutral red (dilute the lab stock 2-3/100 in PBS and filter before using) approximately 1-2 ml/well, distributed over the surface of the methylcellulose. This will diffuse to cover the monolayer and allow visualization of the plaques.
- Count plaques. The neutral red is toxic to the 3T12 cells, so the plaques should be read less than 24 h after addition of the dye.
- Tape and double bag the plates and autoclave.
- List troubleshooting tips here.
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Relevant papers and books
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