I was using MDCK cells to grow Influenza H1N1 in 12-wells plates. Cells was seeded 1 x 10^6 cells/well and left overnight in media without serum. Cells were then infected with H1N1 at moi of 0.1 and virus titre was tested by HA assay after 24 hrs, and 48 hrs.
Trypsin was added during infection.
Under the microscope, the cells was achieved ~80% cytopathic effect after 48 hours. However, no titre was determined by HA assay (all negative HA) either after 24 hours or 48 hours.
Is there any mistake happening here?
In my point of view, first, I guess its because of the temperature during harvesting, virus was harvested at room temperature from 37C cell culture and spin at 4C and then store at -80C freezer for further HA testing.
Second point, was the virus titre too low? or was the cells density too low?
Does anyone here working in the same field or experiment with me before?
What strain of H1N1 and what species of RBCs? Not all flu strains have the right sialic acid preference for a given type of RBC. Also, the HA assay is probably the least sensitive routine titration method for flu. If you used an infectivity-based assay you might find you have some replication.
paul
Hi
1- check the HA method you apply
2- The number of virus passage on cell cultural is crucial for adaptation and high titre
What strain of H1N1 and what species of RBCs? Not all flu strains have the right sialic acid preference for a given type of RBC. Also, the HA assay is probably the least sensitive routine titration method for flu. If you used an infectivity-based assay you might find you have some replication.
paul
What is the concentration of TPCK-treated trypsin?
Too much TPCK-treated trypsin will destroy the cells. You may see something like cytopathic effect, but actually it was because of the TPCK-treated trypsin.
You can also treat the virus with TPCK-treated trypsin during the infection (at 60 minutes, 37 C) and then add the TPCK-treated trypsin to the medium. For infection, it is better to add BSA fraction-V to the medium as a substitution of FBS. Otherwise, your cells will be sick/dead.
In my experience, it might be something with the trypsin and the concentration used. If the virus you are using is the H1N1pdm it normally grows very well in MDCK cells at titers between 1:16 to 1:32 after 48 hrs, detectable by HA.
I recommend that if you repeat the infection, a couple of wells without virus only media and trypsin, will tell you if the concentration of trypsin is affecting the cells.
Is the virus stock you are using, detectable by HA?
I would recommend adding FBS to the cells During growt. for infection wash cell 3 times with PBS (without supplemental Mg). Add virus in growth medium and replace FBC by BSA. Using a mock infected well to exclude any negative effect of Trypsin.
If you have doubts concerning your HA test Check the procedure using a virus with known HA titer In parallel wirh your Stock Virus. In Order to minimize a potential influence of binding preference you might use turkey RBCs which have a broad bindin. Capacity.
Trypsin is added during infection with Rotavirus, this aspect could be useful to you. Good continuation
Maybe you are adding too much virus? Try infecting with a lower dose, allowing the virus to replicate in the cells. Plaque assay to quantify virus titers might be preferable.
My question is, Is it the correct CPE you are viewing? Sometimes the way we inoculated determines the out come.
What percentage of confluence were your MDCK cells when you inoculated also matters.
In this case the temperature should not be a problem unless you freeze thaw your virus supernatant. Some times the HA assay is not sensitive enough to titre all IAV strains. I would suggest you to titrate using plaque assay.
Several parameters are to be considered What level of serum do you use for growing MDCK cells ? in the case where it is 8 or 10%, depriving them of serum all night can make them easily detachable from the support, in this case they should be gradually adapted to grow with small amounts of serum.
Serum inhibits the action of trypsin, in its absence the action on cells is more violent and promotes the detachment of cells that could be confused with a PCE. What is the image of the cells control in your case ?
Is 48 hours enough to have a multiplication of the virus and its release in the supernatant ? Try to break the cells by freezing / thawing the resulting supernatant may be higer in particules and eaisy to detect.
CK cells won't survive in FBS-free serum for long. Cells start to die 36-48 hpi. My guess is that what you are seeing is cell death and not CPE. Thus, you have high number of dead cells and low titer. The solution for this is what was suggested by Jan Baumann.
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