Hi, I'm 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, using nonserum DMEM, and shaking every 15 mins during virus infection incubation (1h at 37C and 5% CO2)
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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