Testing Out Making Fly Extract From innubila Flies

I want to try making fly extract to supplement my primary cell cultures to help them grow. What I did roughly follows the methods in this paper, however there are some changes.

  • Prepped flies by dumping all newly emerged flies that I could spare onto CO2 and waiting for them to sleep
  • Then I weighed them on the portable scale, but they weighed less than a gram so it did not show up. I’m supposed to start with 2g so this was not a good sign
  • I put the flies on ice and weighed them in the sensitive scale in the lab, it said they were 0.12-.2g of flies
  • Because that was all the flies I had, I went ahead to try to make the extract anyways
  • I kept the flies on ice
  • Made a 10mL aliquot of Schneider’s medium and kept it on ice
  • I am supposed to use a blender to mix up the flies and medium, and then a pestle grinder, but I didn’t think there was enough liquid to use the blender
  • Also, we don’t have the “right” size pestle grinder, so I decided to use the 7mL Weaton glass dounce homogenizer instead
  • Even though I didn’t get 2g of flies, it was still a lot of flies to grind up, so I did the grinding in 3 sets
  • I added 2mL cold medium to the homogenizer and about 1/3 of the flies
  • Homogenized for ~5 minutes, it was hard to homogenize them, there were so many flies but after a while they seemed to be pretty broken up
  • “Rinsed” the debris down with another 1mL of cold medium
  • Pipetted out the homogenate into 1.5mL tubes
  • Repeated homogenization 2 more times for the rest of the flies. The last time had fewer flies and was easier to homogenize them completely. I used up all of the 10mL of medium and made 8 1.5mL tubes
  • Centrifuged the tubes in the fridge for 15 minutes at 1,500rcf
  • Transferred the supernatant to new 1.5mL tubes
  • Incubated tubes at 60 degrees C for 5 minutes in the heat oven
  • Centrifuged tubes in the fridge for 90 minutes at 1,500rcf. Our small centrifuge that fits in the fridge only goes for 30 minutes, so I did 3 30 min cycles
  • Used the syringe filter with a 0.22um filter to push the supernatant from the tubes through the filter into a new 15mL tube
  • This resulted in ~8.5mL of liquid that I labeled “D. innubila fly extract” and froze in the -20