An In Vitro Assay to Examine the Antibody-Dependent Enhancement of Viral Infection
Transcrição
Begin by thawing the serum samples at room temperature. When thawed, transfer 100 microliters of each serum sample to a sterile tube and heat-inactivate for 30 minutes at 56 degrees Celsius.
Next, make 10-fold serial dilutions of the serum sample ranging from 1-to-1 to 1-to-1,000 in cold RPMI-10. Then transfer 10 microliters of each diluted serum sample to the wells of a sterile 96-well V-bottom plate.
Next, thaw dengue 1, 2, 3, and 4 reporter viral particles in a 37 degrees Celsius water bath, ensuring a volume of approximately 170 microliters of each RVP per serum sample. Once thawed, immediately transfer the tubes of RVPs to ice. Then pipette 10 microliters of RVPs into the wells containing serum sample dilutions into the no-serum control wells. Mix thoroughly by pipetting up and down five to 10 times.
Transfer the 96-well V-bottom plate to an incubator, and incubate for an hour at 37 degrees Celsius in the presence of 5% carbon dioxide. While the 96-well V-bottom plate is incubating, clean the surface of the biosafety cabinet with 70% ethanol and run the UV light for 15 minutes.
Then, remove a fully confluent T75 flask of K562 cells from the incubator. Mix the cells well using a sterile 5-milliliter pipette, and transfer 5 milliliters to a sterile 15-milliliter conical tube. Next, remove 10 microliters of cells from the tube. Mix with 10 microliters of trypan blue and load into a hemocytometer. Count the cells and calculate the total number of cells. After centrifuging the cells at 1,200 x g for 10 minutes, decant the supernatant and resuspend the cells in warm RPMI-10 at a concentration of 80,000 cells per 30 microliters of media.
After removing the 96-well V-bottom plate from the incubator, transfer 30 microliters of K562 cells to each well of the 96-well V-bottom plate and mix thoroughly by pipetting up and down five to 10 times. Then, return the plate to the incubator for one hour.
Following the incubation, centrifuge the plate at 1,200 times g for 5 minutes. After centrifugation, decant the media from the wells by turning the plate upside down into a container containing 10% bleach. Then, wash the cells in each well by resuspending them in 125 microliters of warm RPMI-10 and centrifuging as before.
After decanting the last wash, add 100 microliters of warm RPMI 10 to each well. Mix up and down with the pipette, and incubate the plate for 48 hours at 37 degrees Celsius in the presence of 5% carbon dioxide.
Two days, later remove the plate from the incubator and transfer it to a biosafety cabinet. Using a multichannel pipette, mix the contents of each well and transfer to a 96-well U-bottom plate. Rinse each well of the 96-well V-bottom plate with 100 microliters of 1% paraformaldehyde in PBS, and transfer the remaining cells in PBS to the respective wells in the 96-well U-bottom plate. Mix thoroughly using a multichannel pipette.
Cover the plate with aluminum foil and let it sit in the incubator for 30 minutes to fix the cells. Prepare the flow cytometer by running unstained K562 cells to calibrate the side and forward scatter settings using the FL1 channel for GFP fluorescence. Then, acquire 30,000 to 50,000 cells from each sample.
