PoPLaR: BioMethodology - Improve Cell Survival

• Reduce the irradiation time
  1. Gain an accurate understanding of how long HeLa cells can withstand vertical
     • Current limits are that they can survive 20 minutes but not ~1.5 hours
  2. Being aware of the issue
  3. Reducing the time between shots from 20s to 5s
     • This would bring the irradiation time from roughly 16 mins to roughly 4 mins
  4. Not irradiating RCF in the same carousel

• Dealing with potential causes: Temperature, CO2, lack of media, weak cell line
  5. Heat the carousel in which the cells are placed
     • Can this be done by placing the carousel in the incubator as well?
  6. Keep the carousel rotating so that the cells are consistently wet
  7. Change the cell dish lid so that more media can be added through a second hole
     • Would also help regulate temperature
  8. Change the cell line from HeLa to the super-resistant ones used in Marie's lab
     • Requires testing the cells at Birmingham and repeating the experiments
     • Not certain it will work

• Equipment factors
  9. Place a thermostat in the incubator to check that it is not too warm
     • Robbie has said he will build a monitor using a Raspberry Pi
  10. Use Marie's lab
      • Need to inform Marie of when so that she can book in the incubator
      • Can do X-ray comparison here
      • Bigger fume cupboards, so 2 people could work at once and reduce the load
      • Comet analysis
      • Easier day-to-day observation

Personal Thoughts

There are two things we do not know. The fastest we can irradiate cells and the time cells can survive vertically. I think if we do 2, 3, and 4, we can get the time the cells are vertical for to near 30 minutes. I think Robbie estmated that the externals including the transport, time to rotate carousel, mounting time, lock-up/prep time and post-irradiation checks take between 15-25 minutes. 3 reduces the irradiation time from 16 to 4 minutes, and 4 would then reduce that to 2 minutes, so then the externals are the biggest factor. A conservative estimate puts the time between 20-30 minutes, so it is possible that 2,3 and 4 are all that are needed to deal with the cell survivability issue.

However, other factors may cause these to change, such as needing to take more shots, so I think it would be sensible to test how long the cells can last vertically (1) and see how much leeway we have.

If there is no leeway, that is when I would start considering 5,6,7 and 8 (probably in that order?).

Either way, a dry run is necessary before more irradiations can be done.

Finally, I would definitely install a thermostat in the incubator; a monitor from Robbie would be great, but not essential given Robbie's workload.

I do not know enough about whether to use Marie's lab. If we do not, we need to acquire an inverted microscope and a multi-chamber haemocytometer.

---