I'm having a problem with this model because the virus isn't causing plaque lysis in the cell monolayer. To titrate, I'm using a semi-solid medium (carboxymethyl cellulose 1,5% plus 1,5% of FBS) over 5 days of incubation. Could be something wrong with the cells or maybe with the semi-solid medium?
Make sure you allow your CMC to cool a bit before you put it on the cells, as keeping it warm in a 37ºC waterbath makes it a bit too hot for the cells sometimes (even though BHKs are kept at 37ºC in an incubator). How much overlay do you put on per well? Also I used 4% autoclaved CMC diluted (1:1) w/ freshly mixed 2% FBS with serum-free media (MEM? DMEM? whatever it was). Make sure your virus is kept on ice when you aren't using it and only make the dilution series right before you add it. Are you using a 12-well plate with 6 dilutions in duplicate? Also sometimes some serotypes don't work well with the plaque assay, and some genotypes within each serotype have different plaquing/lysis phenotypes since DENV isn't a proper lytic virus (cell death and the plaques we see are caused by cellular infection, but normal DENV virions are released through export and not cell lysis from mechanical tension). Allow the assay to incubate for a longer time, and as long as your cell monolayer is not overgrown and coming off the plate, you might start to see plaques come up.
I have never been able to get a plaque assay working for dengue virus. I have found that an immunoperoxidase staining assay is a good way to determine titers. Here is the method that I use from Durbin et al. 2001. ATTENUATION AND IMMUNOGENICITY IN HUMANS OF A LIVE DENGUE VIRUS
TYPE-4 VACCINE CANDIDATE WITH A 30 NUCLEOTIDE DELETION
IN ITS 3-UNTRANSLATED REGION
Vero cell monolayer cultures in 24-well plates. After a 1-hr incubation at room temperature, the monolayers were overlaid with 0.8% methylcellulose
in OptiMEM (Life Technologies) supplemented with 5% FBS. Following incubation at 37C for four days, virus plaques were visualized by immunoperoxidase staining.
Briefly, cell monolayers were fixed in 80% methanol for 30 min and rinsed with antibody buffer (5% nonfat milk in phosphate-buffered saline). Rabbit polyclonal DEN-4 antibodies were diluted 1:1,000 in antibody buffer and added to
each well followed by a 1-hr incubation at 37C. Primary antibody was removed and the cell monolayers were washed twice with antibody buffer. Peroxidase-labeled goat-anti-rabbit IgG (Kirkegaard and Perry Laboratories, Gaithersburg,
MD) was diluted 1:500 in antibody buffer and added to each well, followed by a 1-hr incubation at 37C. Secondary antibody was removed and the wells were washed twice with phosphate-buffered saline. Peroxidase substrate (4-chloro-1-
naphthol in H2O2) was added to each well and visible plaques were counted.
Emerson, BHK cells will not work for dengue virus. BHK cells grow too quickly for this cell line to be used as an indicator strain. I'm attaching our lab protocol. e-mail if you have any questions.
Thank you very much for your input.
Louis, I’m using a 12-well plate with 5 dilutions in duplicate, with CMC medium 60 to 80% of the well volume.
Jeffrey, I´ve been reading read about the immunoperoxidase staining assay. It looks pretty good and easier, but I’ll need to titer all of the serotypes of dengue. So, I think it's more expensive. Do you know if there is an antibody commercially available that can recognize all four dengue viruses serotype that is suitable for this assay?
Rosmari, I seed 100 000 cells/well in a 12-well plate or 400 000 cells/well in a 6-well plate. About my viral stock, I checked for the viral CPE, like syncytia formation in C6/36 cells infected with moi=0,01.
Thanks for protocol Raquel. I’ll try to make the titration with the Vero cell line.
Thank you all again.
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