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 maybe 1s, definitely to 5s
  4. Not irradiating RCF in the same carousel
     • A combination of these two would bring the irradiation time from roughly 16 mins to roughly 4 mins
• 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
      • Much 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 less than 20 minutes. 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 time, then I would go to SCAPA and test 5 and 6. If time allows, might as well test 7 and 8, but this feels like it will take longer for the new lids and the subsequent work of the new cell line.

Either way, a dry run is completely 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 essential.

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.

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