An In Vitro Recombinant Virus Reporter System for the Detection of Viral Infection
Transcrição
Plate HepG2 cells, stably expressing sodium taurocholate, co-transporting polypeptide, or NTCP that are susceptible to HBV infection, and incubate at 37 degrees Celsius and 5% CO2.
One day before infection, plate approximately 5 x 104 HepG2/NTCP cells into the wells of a 96-well collagen-coated plate in 0.1 milliliters of culture medium. Thaw recombinant HPV in a 37 degrees Celsius water bath, until there is a small bit of ice remaining in the vial.
Prepare medium for infection by combining 78 microliters of fresh culture medium, two microliters of DMSO, 10 microliters of 40% PEG8000 in 1X PBS, and 10 microliters of recombinant HPV. Add 100 microliters of recombinant HBV solution to each well of a 96-well plate.
One day after infection, use 300 microliters of PBS to wash the cell three times, to remove the contaminating reporter protein from the virus fraction. Add 200 microliters of culture medium containing 2% DMSO to the infected cells, and incubate for one week or less.
To carry out analysis of the HBV infection, one week after infection, use PBS to wash the infected cells three times. Add 50 microliters of lysis buffer to the infected cells. Rock the culture plate for five minutes, then, centrifuge at 2000 times g for five minutes. Add 50 microliters of the reporter substrate to a luminometer plate, then, add 20 microliters of the cell lysate to the plate, and mix by briefly shaking. Finally, place the plate in the luminometer and initiate reading.
