The overall goal of this procedure is to quantify infectious neuron norovirus particles. This is accomplished by first preparing tenfold dilution of virus inoculum. The second step is to transfer dilution of virus inoculum to a raw 2 6 4 0.7 monolayer and incubate plates rocking at room temperature for one hour.
Next, aspirate the inoculum and overlay with a one-to-one ratio of C plaque aros and two XMEM and incubate the plates for 48 hours in a tissue culture incubator. The final step is to overlay with neutral red staining solution and incubate the plates for one to three hours in a tissue culture incubator. Ultimately, the neutral red staining solution is aspirated carefully without removing the arose overlay and plex accounted using a light box.
This method of quantifying infectious viral particles can help answer key questions in the norovirus field, such as the amount of infectious particles shed in the feces, or which tissues support infection. Generally, individuals new to this method will struggle with maintaining healthy, viable cells throughout the assay. Visual demonstration of this method is critical as the plaque assay for mirror neurovirus can be difficult to grasp for people without prior experience.
The successful completion of this assay is a cumulative series of steps that require the right amount of cells, the correct temperature and precise handling in order to readily observe plaques. First, examine the cell monolayer under a light microscope to check the morphology of the cells. The majority of the cells should appear round.
Do not let the cells overgrow since they typically will not form plaques to massage a nearly cofluent 175 centimeter square tissue culture flask of raw 2 6 4 0.7 cells for the murn neurovirus plaque assay aspirate the media from the flask and add 10 milliliters of fresh DMEM 10 to the cells. Next, scrape the cells from the bottom of the flask with a cell scrapper and use a 10 milliliter pipette to aspirate and then expel the cell suspension against the bottom of the flask. Repeat this action at least three times to break up any clumps of cells.
To suit a stock flask. Remove one to two milliliters of cell suspension and deposit it into a fresh 175 centimeter square flask containing 33 to 34 milliliters of DMEM 10. Incubate the flask in a tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide for two to three days and massage again.
Once complement to seed the plates for the assay, adjust the density of the cell suspension to one times 10 to the six viable cells per milliliter in DMEM 10 and add two milliliters of this suspension to each well of a 3.5 centimeter diameter.Six. Well plate cover the plate. Then immediately rock the plate by hand 10 times to distribute the cells evenly in the wells.
Do not swell the plate as this will cause the cells to cluster in the center of the well. Then place the plate in a tissue culture incubator for a minimum of four hours or ideally overnight for the cells to attach virus inoculum can be obtained from either MNV infected homogenized tissues or fecal samples from MNV infected mice. If using tissue samples, place a pea-sized piece of tissue in a two milliliter screw cap tube containing sterile silica beads and one milliliter of DMEM 10 and homogenize here.
A magnetizer is used at 6, 000 RPM for one minute. If using fecal samples, homogenize no more than three fecal pellets in the same manner, freeze the homogenized samples at minus 80 degrees Celsius and thaw the inoculum on the day of the plaque assay. Use a repeat of pipette to dispense 1.35 milliliters of DMEM five into each well of a 24 well plate then pipette 0.15 milliliters of the third virus inoculum into the first well of the plate.
Perform tenfold serial dilutions by aspirating 0.15 milliliters of the first 10 to the minus one dilution and transferring it to the second well of the plate to dilute 10 to the minus two. Then repeat the process with the dilution from the second well to dilute to 10 to the minus three. Note that further dilution up to 10 to the minus nine may need to be made depending on the sample.
Once the serial dilution are prepared, label the plates of 60 to 80%confluent raw, 2 64 0.7 cells with the sample names and the dilution being used. Then remove all the media from one of the plates by aspirating or flicking out and add 0.5 milliliters of a diluted sample to a well. Repeat with a duplicate well and then proceed to the next lower dilution.
Once all three dilution have been added to one plate in duplicate, tilt the plate back and forth to ensure that all cells have been covered. Repeat the procedure with the next plate of cells. Once all of the plates have been treated, place the plates on a rocking apparatus set at around 18 oscillations per minute for one hour at room temperature, microwave a bottle of previously prepared and or enclaved aros until melted.
Then place the molten aros in a 42 degrees Celsius water bath to equilibrate to temperature. Next, mix the molten agros with sterile two XMEM media in a sterile bottle at a one-to-one ratio. Place the bottle containing this overlay solution in a 37 degrees Celsius water bath until ready to use.
At the end of the one hour incubation, aspirate the inoculum from each well and slowly add two milliliters of the overlay solution to each well, ensuring that the pipette tip is placed against the wall of each well. To prevent dislodging the cells cover the plates and allow the overlay to solidify for approximately 10 minutes of room temperature. Then transfer the plates to the tissue culture incubator for 48 hours.
Following the incubation period, check the plates for the presence of plaques. If no plaques are visible, return the plates to the incubator and then check again after four hours. Repeat the procedure if necessary, but the incubation should not exceed 72 hours.
First, prepare the neutral red staining solution. Then add two milliliters of neutral red solution to each well for this protocol, the aros plug does not need to be removed. Incubate the plates at 37 degrees Celsius for one hour.
After the incubation time has elapsed, check the plates for the presence of plaques while keeping the neutral red solution in the wells. If no staining is apparent, incubate the plates for another hour for a maximum of three hours. Once plaques are visible, aspirate the neutral red solution, ensuring that the arose plug is not disturbed, and then proceed to counting the plaques.
Examine the plates and find the dilution where the plaques are clearly separated. Place the plate on a light box and count the plaques while marking a dot on counted plaques. To avoid duplicate counts.
Then repeat the count on a well with a different dilution of virus and calculate the viral titer in plaque forming units per milliliter. According to the instructions in the written protocol, this representative image shows a monolayer before infection. The raw 2 6 4 0.7 cells were cultured overnight and imaged under a light microscope a 20 times magnification.
This image shows the same monolayer after formation of plaques. Cells were stained with a 0.01%neutral red solution after 48 hours of infection and visualized under a light microscope of four times magnification. Roman numerals one, two, and three indicate three visible plaques.
This image shows a plate at the end of the plaque assay protocol with duplicate wells of three tenfold dilution wells labeled with Roman. Numerals one and four correspond to the 10, to the minus one. Dilution two and five correspond to the 10, to the minus two dilution and three and six correspond to the 10 to the minus three dilution.
The viral titer of the sample is indicated below. While performing this procedure, it is important to remember to change pipette tips between dilutions to avoid cross contamination between samples, equilibrating the solutions to the correct temperature, and not to let cell mono layers dry out. After watching this video, you should have a good understanding of how to perform a plaque assay and quantify mi neurovirus infectious particles.
Don't forget that when working with Mira Norovirus in a biosafety cabinet, precautions such as wearing gloves and gown should always be taken while performing This procedure and waste should be properly disposed of following the guidelines established by your local biosafety committee.
