Attempting Whole Fly Cell Culture with Kent (watching) 2021 10 05

Starting Notes

  • Warm up all media to room temp inside the TC hood before use
  • Serum, 420 medium, trypsin, and antibiotics
  • When changing the red trash bag in the TC room, double bag and twist tie, needs to be autoclaved
  • Regular tips are sharps and go in the glass disposal box
  • 50mL tubes are in the drawer by the door
  • Using 2 cell startiners: 400um green and 100um yellow

Making Medium

  • Made 3 50mL conicals: 1 with serum and 2 without
  • Labeled tubes
    • 420 medium, 10% serum (if applicable), and antibiotic abbreviations
    • Penicillin, streptomycin, Amphotericin B (antifungal), and gentamicin
  • 10% serum medium
    • 5mL serum
    • 44.5mL 420 media
    • 500ul 100X antibiotics (P, S, AB)
    • 50ul gentamicin
  • Wash medium, no serum
    • 49.5mL 420 media
    • 500ul 100X antibiotics
    • 50ul gentamicin
  • Used serological pipettes and micropipettes. MP tips are kept in the TC hood.
  • If tips (any) touch anything but the liquid they are sucking up, you must throw it out (red bag bucket) and get a new one
  • Cap and invert to mix conicals
  • Keep serum media separate in the hood so you don’t mix them up
  • Put media components back into the fridge

Whole Flies - Homogenizing

  • Flies were bleached (10%) and are in PBS (2 tubes, same flies)
  • Homogenizing too (kept in 4055) uses plastic pestles that have to be autoclaved before use
  • When adding pestle to the tool, try not to handle it. If needed to, don’t touch the tip and the beaker with the rest will need to be autoclaved again for the remaining pestles
  • Homogenized both set of flies (separate pestles though) for about ~30-40 seconds each
  • Liquid got dark, not all the pieces of the flies broke up/went into suspension
  • Pelleted homogenate in the 1.5mL centrifuge: 1 min at 13,000 rpm
  • Set up vacuum for aspiration:
    • Plug lining to the yellow vac valve in the other hood (need to plug both ends of the line in)
    • Turn on vac not to high
  • Added a pasture glass pipette to the vac line inside the TC hood
  • Aspirated out supernatant from each pelleted homogenate
  • Got a screw-cap siliconized glass tube
  • Added 500ul trypsin to the pellet in the 1.5mL tubes
  • Resuspended the pellet by pipetting
  • Pipetted mix into the siliconized tube (chunks and all)
  • Repeated for second tube with a new tip
  • Added 8.5mL fresh trypsin to the glass tube
  • Inverted to mix
  • Used program 2 in the centrifuge near the TC room to pellet (400rpm for 3 min)
  • Used same type of tube with DI water for balance
  • Used pasteur pipette and vac to suck out supernatant
  • Added 10mL new trypsin to the glass tube
  • Inverted to mix
  • Placed horizontally in the 27 degree incubator for ~15 min

Filtering and Flasking

  • In sterile erlenmeyer flask (autoclaved) add new yellow cell strainer, then green cell strainer on top
  • Use pasteur pipette (not on vac) to suck up trypsin/fly mix from the incubator tube and pipette out through the filter setup
  • The flow through in the flask should have the cells (smaller than 100um)
  • Got a plastic 15mL tube
  • Used a 5mL serological to pipette flowthrough into the 15mL tube
  • Pellet cells: program 2
  • Aspirate out the supernatant with vac and pasteur pipette
  • Use media w/out serum to wash: add 5mL
  • Inverted to mix
  • Pellet again with program 2
  • Aspirated out wash media with vac/pasteur pipette
  • Resuspended pellet with 5mL of media WITH serum
  • Transferred liquid to a 25 cm2 flask (orange lid)
    • Named flask: inubila P1 primary 10/5
  • Put in the 23 degree incubator