Hello,
I am trying to treat A549 cells infected with PR8 and I am trying to determine the best media conditions but in the literature conditions are all over the place. I see 2% FBS, 5% FBS, 10% FBS, no FBS, 0.2% BSA, DMEM, MEM, L-15, etc...
For my experiments I have been infecting cells (1 hr 37C) in serum free DMEM. After this, I replace with 10% FBS F-12 medium and the treatment (usually in 0.3% methanol). I figure using no FBS is not representative of what actually happens in vivo.
Is there any general of thumb for this types of experiments or does it come down to optimizing conditions for every strain and treatment?
Thank you!
Randy,
Thank you for the response. I intend on looking at replication along with gene expression (e.g. NF-kB dependent), reactive oxygen species production, and other components of the innate response.
For experiments solely looking into replication I'll try your recipe (I normally use 2 ug/mL TPCK). I'm just worried a serum free environment would significantly alter the cells I am infecting, but I guess it is a give and take with influenza replication?
Greg
Best not to use FBS and this can inhibit virus replication. Use media (DMEM, BSA, TPCK-Trypsin). Would definitely check concentration of TPCK-trypsin as Randy mentioned, could cause cells to detach. Try titrating TPCK-trypsin with uninfected cells first to see where optimal dose lies which does not cause cell detachment/morphological changes. Then infect and use that concentration. A low dose should be sufficient.
Hi, Randy A Albrecht I'm also using A549 and PR8 H1N1 virus to do plaque assay and I'm facing a problem as my cells always wash off after I fix it using 4% formaldehyde.
Currently, I'm using 2ug/ml TPCK, 0.6% agarose gel, and L15 media as the overlay media, however, the cell always washes off after 48 hours of incubation. I'm wondering if it happened because of the concentration of TPCK that was too high and become toxic to my cells which subsequently cause cells to detach. I don't think there is any problem with the cell concentration as I always proceed with the treatment when it was 90-100% confluence. Is there any recommendation for the concentration of agar and TPCK for plaque assay using A549 cells?
I would appreciate your help as I only have limited time to finish this assay. Thank you!
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