Plating Dinn Cells for DiNV 3-4 Month Experimental Evolution Experiment

We are wondering if DiNV would evolve in the same way if let grow in cell culture for a few months. That could test if it has adapted to cell culture or if some changes we see are just drift.

Goal is to monitor evolution of DiNV fly homogenate in cell culture over ~3 months. I want 3 replicate flasks to get the same inoculum, I want to save the inoculum for sequencing, and I want to take samples of supernatant fluid throughout to check for virus and for potential timepoint sequencing.

Inoculation will happen on Sept 19th, the experiment will end 3 months later on the 19th of December which is a good time to finish because it is too early to be out of town for the holidays. Or it can be extended another month easily.

We have decided not to pass the flasks because there is the concern of the cells not growing or dying if trying to pass them when infected. Or if we use trypsin the cells losing their infection in some way.

Making the flasks

  • Took passage 6 3.1 Dinn cells and poured off the medium
  • Added 5mL of 0.05% Trypsin, spread it around, and poured it off
  • Added another 5mL of 0.05% Trypsin and let the flask sit in the hood for 10-15 minutes, periodically rocking the flask back and forth to release the cells
  • Pipetted off the 5mL of cells into a 15mL conical
  • Spun down the conical for 2 minutes at 200rpm
  • Poured off the supernatant from the cell pellet
  • Resuspended the pellet in 5mL of 10% FBS 4% mushroom extract Schneider’s medium
  • Made sure solution was resuspended well, and took 1mL and added that to 3 new flasks labeled A, B, and C
  • Took 3 replicates of 50ul of the cell suspension and put it into 1.5mL tubes labeled 1, 2, and 3
  • For both of these, I tried to make sure the cell solution was completely mixed up when pipetting
  • Added 5mL of fresh medium to each of the flasks and placed them in the 23C incubator to settle and grow for about a week
  • The 1.5mL tubes were frozen at -20 and DNA extracted at a later date to check for presence of virus
tube number day sampled sample volume
1 day -6 50ul
2 day -6 50ul
3 day -6 50ul