I'm trying to propagate a H1N1 strain in DMEM plus 0.2% BSA and 1 ug/ml TPCK-trypsin but mock treated cell (uninfected) died after 24 h. I repeated twice and the same phenomenon happened. This never happened before when I used MEM. Does DMEM + BSA toxic?? However, MDCK is growing well in DMEM + 10% FBS..Thanks!!
Hi.
You will need to exclude TPCK-trypsin from medium. You can add the TPCK-trypsin but only with virus (H1N1) when MDCK grown up to 70-80 % of monolayer.
The trypsin does sound like the most likely reasons for cell death, particularly in such a short time frame. If you still see susbtantial death at a reasonable cell confluency it might be worth titrating the trypsin to see if you can reduce the dose - after aliquotting to produce working stocks there can be some variation in strength between batches.
Your BSA concentration seems rather high. We routinely use 0.3% BSA with 1ug/ml TPCK four our pdm09 infection in A549. We did accidently used 3% BSA once which resulted in significant cell death. For MDCK, we did not find the addition of BSA necesarry. Make sure you wash off your growth media containing FBS from cells at least twice with PBS. Also, a collegue did a comparison DMEM vs. MEM for pdm09 infection in A549 cells to troubleshoot his poor infection level, and found that while MEM resulted in >90% of cells infected by 48hr, only 20% of cells were infected when he used DMEM (by immunostaining). We did not see this difference with other flu strains like X79, or WSN.
I think the nature of the culture medium has an indirect effect on your virus. The choice of medium is better growth of cells that determine a good multiplication of virus. The role of trypsin and the concentration of BSA are reported correctly . The presence of trypsin in the environment could affect the health of cells and therefore the viral cycle.
Make sure you test each lot of TPCK-Trypsin for toxicity prior to use for infection. There are lot to lot differences in trypsin activity
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