A Focus-Forming Assay for Quantifying Virus Titers in the Organs of an Infected Mouse
Trascrizione
Remove the homogenized samples from the minus 80 degrees Celsius freezer and allow them to thaw. With the samples on ice, dilute Zika virus 10-fold into the first well of the dilution plate, and use a multichannel pipette to do 10-fold serial dilutions down the plate, changing tips each time by adding 20 microliters of sample into 180 microliters of growth media.
Change pipette tips between each dilution. Prepare the focus-forming plate by removing the media from the prepared flat-bottom 96-well plate covering Vero cells. To prevent the monolayer from drying out, immediately add 100 microliters of the virus dilution to each well in the Vero plate. Use the same set of tips to add from the lowest to the highest concentration.
Rock plates side to side 2 to 4 times, and incubate at 37 degree Celsius and 5% carbon dioxide for one to two hours. Warm up 2% methylcellulose to room temperature. Then, dilute the 2% methylcellulose solution in growth media at a ratio of approximately 2 to 1. After incubation, add 125 microliters of the methylcellulose growth media to each well of the 96-well plates. Incubate again at 37 degrees Celsius and 5% carbon dioxide for 32 to 40 hours.
To fix the Vero cells, in a biosafety cabinet, add 50 microliters of 5% paraformaldehyde over the top of the methylcellulose layer in each well of the 96-well plates. Incubate for 60 minutes at room temperature. Alternatively, cover the plates with parafilm, and place them at 4 degrees Celsius overnight.
After that, use a pipette to remove overlay and media off cells into a disposable container inside the biosafety cabinet. Wash gently with 150 microliters of PBS per well. Remove PBS from the plates and remove the plate from the biosafety cabinet. Repeat the PBS wash twice. Then, add 150 microliters per well one times FFA wash buffer and leave at room temperature for 5 to 10 minutes to permeabilize the fixed cells.
Next, use primary Zika antibody to detect infection. Prepare the primary antibody 4G2 at a concentration of 1 microgram per milliliter in FFA staining buffer. Flick FFA wash buffer from the plates, and add 50 microliters of the primary antibody in FFA staining buffer to each well. Then, seal the plates with parafilm or plastic film, and incubate overnight at 4 degrees Celsius.
Prepare the secondary goat anti-mouse HRP-labeled antibody at a concentration of 1 to 5,000 in FFA staining buffer. After removing the antibody solution, wash cells three times with 150 microliters FFA wash buffer per well. Remove the wash buffer by flicking into the sink each time.
Stain the cells with the secondary antibody in FFA staining buffer at 50 microliters per well, and incubate one to two hours at room temperature. After that, wash cells three times with FFA wash buffer again, removing the wash buffer by flicking into the sink each time.
Add 50 microliters of the Trueblue substrate into each well. Wait 2 to 15 minutes until spots are fully defined with minimal background. After the spots are visible, wash gently with water using a hand to shield the monolayer from the force of the water running. Tap-dry on paper towels.
Shortly after, use a dissecting scope to count manually the spots in each well or use an automated spot counter. In the immunocapture software, select a dilution with easily distinguished foci between 20 to 200 per well for each sample, and calculate titer in focus-forming units per milliliter using the average of duplicate wells.
