Second Updated Protocol For Preparing Drosophila Eggs To Primary Cell Culture

This is the protocol starting from a plate already with eggs laid

Steps

Set up

  • Make 40% bleach solution (this is for 2 uses)
    • 8mL bleach
    • 12mL 1X wash
  • Turn on the UV in the TC hood for 10 minutes (leave door closed)
  • After the 10 minutes, turn on the blower, fluorescent light, and open the hood
  • Pour a small beaker with some 70% ethanol for washing forceps and paintbrush
  • If needing to make new media, do this in the TC hood after the UV has finished:
  • 1 50mL conical of 20% FBS media
    • 10mL of serum
    • 39.5mL of Schneider’s medium
    • 500ul of 100X antibiotics (Penicillin, Amphotericin B, and Streptomycin)
    • 50ul of 50mg/mL Gentamicin
  • 1 50mL conicals of media without FBS (0%)
    • 49.5mL Scheneider’s medium
    • 500ul of 100X antibiotics (Penicillin, Amphotericin B, and Streptomycin)
    • 50ul of 50mg/mL Gentamicin
  • Let the medium warm up to room temp for use

Fly Room

  • If doing a second day of laying, bring the second apple juice and yeast plate to the fly room
  • Set up CO2: unscrew tank if not on, plug in hose and open valve
  • Tap fly cage onto the CO2 plate until all flies are asleep
  • Take out red cap and plate and cover the plate immediately
  • If doing a second day of laying:
    • Put in new yeast plate to the red part and re-cap cage
    • Rotate the cage horizontally until the flies wake up
    • Set up the cage for overnight
  • If flies are done:
    • If not keeping flies, dump them out into the morgue
    • If keeping flies, separate them out into 3 equal groups on the CO2 plate and add them to newly labeled vials
  • Turn off all CO2
  • Take plate covered back to 4012

Filtering: At the Lab Bench

  • Pick off dead flies with tweezers and put in ethanol
  • Set up the filter rig: autoclaved erlenmeyer flask, 100um filter, 400um filter, then the funnel on top
  • Squirt water into the plate and begin mixing up the yeast with a paint brush, mix until as best you can get homogenous
  • Tip over the plate into the funnel and squirt water to wash the liquid down
  • Brush and rinse at least 2 more times to wash all the yeast and eggs out of the plate
  • Take off the funnel and rinse the green filter with water
  • Take of the green filter and rinse the yellow with 1X wash for close to 2 minutes
  • Flip the yellow filter over into a new 50mL conical
  • Serologically pipette 7.5mL 40% bleach into the filter to wash out the eggs into the conical below
  • Use a pasteur pipette (pp) to transfer the 7.5mL to a siliconized 10mL tube
  • Let the tube sit for ~10 minutes: periodically flipping upsidedown to mix so the eggs aren’t settled at the bottom and become anoxic for too long
  • Centrifuge for 3 minutes at 400rpm

Cell Culture Preparation: In the Tissue Culture Hood

  • media are at room temp
  • Set up vacuum: attach tubing to the hood and to the vac in the chemical hood. Turn on the vac very far then back down
  • Use a pp and the vacuum to aspirate off the bleach from the 10mL tube, avoid the pellet
  • Add 7.5mL of 0% FBS medium
  • Centrifuge 1 min 400rpm
  • Aspirate off the liquid with a pp
  • Add 7.5mL 0% FBS medium
  • Centrifuge 1 min 400rpm
  • Aspirate off the liquid with a pp
  • Add 10mL 0% FBS medium (3rd wash)
  • Centrifuge 1 min 400rpm
  • Aspirate off the liquid with a pp
  • Add in 2mL of 20% FBS medium
  • Unwrap dounce mortar and put in a separate rack
  • Use app to transfer all the liquid from the 10mL tube to the dounce (here, many of the eggs will have stuck to the side of the 10mL tube, use the pp to scrape off the eggs into the liquid. This can take a while. Then add the liquid to the dounce)
  • Let the eggs settle to the bottom of the homogenizer
  • Use the same pp to remove ~1mL of liquid from the homogenizer (should not remove any eggs)
  • Unwrap the pestle from the foil and homogenize very briefly: push down and twist for ~5 seconds, lift up, then do again. Should not over homogenize, just until you don’t see any big pieces/eggs left
  • Lay the pestle back down on the clean autoclaved foil
  • Transfer the liquid to a new 10mL autoclaved tube with a pp, making sure you pipette mix a little to make sure you get all settled cells
  • Add 1-2mL more 20% FBS medium to the 10mL tube
  • Centrifuge 10mL tube 2 minutes at 200rpm
  • There should be a pellet of cells visible after this
  • Aspirate off the supernatant with a pp
  • Then resuspend the cells with 5mL of 20% FBS medium
  • Set up the TC flasks. If there were very few eggs, only use one flask. If there were an ok amount of eggs, use 2 flasks. If there were a lot of eggs, use 3 or 4 flasks
  • In one of the flasks, add 5mL of 20% medium per number of flasks - 1 (if you have 3 flasks, add 10mL fluid because you already have the other 5mL in the tube)
  • Pippete mix the tube of medium and cells to break up any clumps
  • Transfer the medium and cells from the tube to the flask with fluid
  • Pipette mix the flask to distribute the cells throughout the liquid
  • Transfer out 5mL of fluid to the remaining flasks
  • Place flasks in the 23 degree incubator horizontally. Let them settle for a bit before looking in the scope

Cleanup

  • Put all media back into the cell culture fridge
  • Put all used pasture pipettes in the glass disposal box
  • Rinse out the plastic beaker for used pipettes with tap, then distilled water, then spray with ethanol and leave to dry in the TC room
  • Rinse out the dounce mortar and pestle with tap then distilled water. Leave to dry for a few hours then wrap in foil for autoclaving. Wrap so that when unwrapping, the mortar comes out first, then the pestle
  • Rinse out the 10mL tubes with tap, then distilled water. If there are eggs stuck on the sides, add a small droplet of detergent, shake up the tube to mix, then rinse many times. Let dry for a few hours then loosely cap, and tape for autoclaving
  • Rinse out flask for filtering with tap, then distilled water, dry for a few hours, then foil for autoclaving
  • Rinse out the beaker with ethanol with tap, then distilled water, dry for a few hours, then foil for autoclaving
  • Clean up all wrappers of serological pipettes
  • Turn off and un-plug the vacuum setup
  • Spray a paper towel with ethanol and wipe down the TC hood workspace
  • Autoclave everything so that it is ready for the next day