An Assay to Quantify the Binding of Human Norovirus VLPs to Intestinal Bacteria
Trascrizione
After preparing the reagents, antibodies, and bacteria as described in the manuscript, assemble all the materials in a BSL II biosafety cabinet.
Virus-like particles for human norovirus are listed as BSL II pathogens, and all work performed using VLPs should be conducted in a certified biosafety cabinet.
Add 10 micrograms of human norovirus VLPs to each tube of bacteria, and mix thoroughly by pipetting. Incubate the tubes for 1 hour at 37 degrees Celsius with constant rotation. After incubation, centrifuge the tubes at 10,000 times g for 5 minutes.
Aspirate and discard the supernatant and resuspend the bacterial pellet in 1 milliliter of PBS. After repeating the wash steps once, centrifuge the tubes again at 10,000 times g for 5 minutes. Discard the supernatant and resuspend the VLP bacteria pellet in 150 microliters of 5% blocking buffer.
A common error that occurs is that tubes are swapped and the wrong treatment is added. To prevent this, arrange all tubes in lines that correspond to the correct treatment, and add one treatment all at once to the tubes.
For each bacterial sample, prepare 50 microliters of diluted human norovirus G2 antibody. Using 5% blocking buffer, dilute the antibody 1 to 125 for E. coli case samples. Prepare 50 microliters of diluted isotype antibody for each bacterial sample using the same dilution ratios. Divide each attachment assay sample into 350-microliter aliquots by transferring them into clean 1.5-milliliter centrifuge tubes.
To create unstained controls, add 50 microliters of blocking buffer to the first aliquot from each bacterial sample. For the stained samples, add 50 microliters of the G2 antibody dilution to the second set of aliquots. Create the isotype controls by adding 50 microliters of the diluted isotype antibody to the third set of aliquots.
Incubate all the samples on ice and in the dark for 30 minutes. Then, centrifuge all the samples at 10,000 times g for 5 minutes. Discard the supernatants and resuspend each sample in 150 microliters of FCSB. Again, centrifuge all the samples at 10,000 times g for 5 minutes. Again, discard the supernatants and resuspend the samples in 100 microliters of FCSB.
After centrifuging the samples one final time and discarding the supernatant, resuspend the samples in 150 microliters of FCSB. Transfer each sample to a tube containing 400 microliters of FCSB for a total volume of 550 microliters. Store the samples at 4 degrees Celsius until they are analyzed by flow cytometry.
