I am trying to determine viral titer and we do not have MDCK cells. My logic tells me influenza replicates in lung cells and not kidney cells and yet MDCK has been established for many years. Why is this so? Can other cell types (i.e. lung cells) be used for plaque assay?
for the propagation of influenza viruses quite some studies have been done.
Historically influenza viruses were propagated in hens' eggs however, this is quite difficult for human influenza viruses.
best results are booked indeed with MDCK cells for both influenza A and B
VeroE6 also works but far less efficiently than MDCK another option could be PERC6/
For a plaque assay to work properly it is important that a single infectious particle gives rise to a microscopic area of cytopathology and thereby measuring infectivity. Therefore, it is critical the cell line used is susceptible for the virus your testing.
MDCK is better cells lines for influenza virus, A549 Violation of tertiary structure of a number of proteins influenza virus, for examle HA. Also can use CACO-2 cell culture (carcinoma from colon)
A549 cell line was established from lung adenocarcinoma tissue and the signal transduction is totally different from normal airway epithelial cells and alveolar epithelial type I and II cells. In particular, A549 is famous for the silencing CD44 expression, which is essential for the binding to ECM components such as hyalronic acids. To better understand the Influenza biology, the glycobiology is essential, so that CD44 expression should be positive in the cell lines in the research of interest.
Thank you so far everybody! I'm trying to be able to test influenza in a cell model that closely resembles the lung and be able to measure titer. MDCK cells are great but influenza does not naturally replicate in the kidney. Perhaps RT-PCR will be more reliable than a plaque assay.
Go,
Do you think A549 cells are a relevant model to study influenza? They are common lung cells used in many papers to study various aspects of disease pathology.
Greg
Pragmatic reasons too - if you're working with trypsin-dependent strains of influenza then you need to use a cell type that doesn't mind being overlaid with ~ 1 ug/ml trypsin. MDCKs are perfectly happy with this, whereas A549s are less so.
Re the RT-PCR suggestion - yes, but be aware you're not measuring infectivity. There are times this will matter (comparing mutants that have different particle:infectivity ratios for instance).
Paul
Thank you everybody! I think I will try and get a hold of some MDCK cells soon and start some experiments.
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