Making 8mL of Fly Extract from D. innubila for Ovary Primary Cell Culture
Followed a protocol similar to this paper without the blender step.
- Chilled the centrifuge in the 4C
- I had two collections of D. innubila flies that I did not need, they were frozen at -20 until use
- I weighed the frozen tubes of flies after taring an empty tube, and I had about 1.5g of flies (protocol says to use 2g)
- Chilled ~10mL of Schneider’s medium and the autoclaved glass 7mL dounce homogenizer on ice
- Homogenized the flies in 3 batches, using 3mL of medium and about 1/3 of the flies
- These are super hard to homogenize because the amount of flies is just huge. They don’t move up and down the homogenizer well. But I was able to crush them well anyways
- Moved the liquid/fly mix with clipped pipette tips to 1.5mL tubes in 1mL aliquots
- Centrifuged for 15 minutes at 4C, 1,500g
- Transferred supernatant to new 1.5mL tubes
- Incubated teh tubes in the heat block at 60C for 5 minutes
- After this the tubes looked kind of opaque, like a precipitate was there
- Centrifuged for 60 minutes at 4C, 1,500g
- Note this was supposed to be for 90 minutes but I ran out of time
- There was a large precipitate pellet that was whiteish at this step, seemed good
- Passed the supernatant through at 0.22um syringe filter into at 15mL tube and froze it at -20