Second day of Myd88 laying and cell culture prep

Collecting Eggs and Filtering

  • Took out 420 medium with and without FBS from the fridge and placed in the hood to warm up to room temp
  • Opened hood, turned on blower and UV light for 10 minutes NOTE mediums should not be in the hood while the UV is on, I made this mistake and put them on the bench after a few min in the hood
  • Went to the fly room to collect eggs
  • Plugged in CO2 hose, turned on valve
  • Tipped over cage onto the CO2 plate and tapped until all the files were asleep
  • Took out the red end of the cage and the plate
    • Covered the plate immediately
  • Dumped the sleeping flies into the morgue
  • Took plate to 4012
  • Made a small beaker of 70% ethanol, and picked off dead flies from the plate into the beaker
  • Set up the filter rig: autoclaved erlenmeyer flask, 100um filter, 400um filter, then the funnel
  • Squirted water into the plate and began mixing up the yeast with a paint brush NOTE I did not make the 50% bleach solution before starting, Kent did it for me while I was filtering. 10mL bleach and 10mL 1X wash
    Also note I was using the paint brush at a flat angle but it might be better to use an upright angle
  • Tipped over the plate into the funnel and squirted water to wash the liquid down
  • Not all the yeast was gone so I brushed and rinsed two more times
  • Took off the funnel and rinsed the green filter with water
  • Took of the green filter and rinsed the yellow with 1X wash for at least a minute
  • Flipped the yellow filter over into a new 50mL conical
  • Used a serological pipette to add 10mL of the 50% bleach solution to the yellow filter to wash out the eggs
  • Immediately used a pasteur pipette and pipetter to transfer the 10mL bleach and eggs to a glass siliconized 10mL tube
  • Let the tube sit for 5 minutes
  • Centrifuged the 10mL tube for 3 minutes at 400rcf, there was a good pellet of eggs

In the Tissue Culture Hood

  • Set up the vacuum line
  • Used vac and silitionized pasteur pipette (SPP) to suck out the supernatant
  • Added 10mL 420 medium without serum to the 10mL glass tube and inverted
  • Centrifuged the 10mL tube for 3 minutes at 400rcf
  • Repeated the wash twice:
    • Aspirated off the liquid with SPP
    • Added 10m: medium without serum
    • Spun 3 min 400rcf
  • Aspirated off the liquid until ~200ul left
  • Added 2mL of 420 medium with serum
  • Unwrapped the dounce, placed the glass mortar in a tube rack NOTE Next time I will need a separate rack for the dounce and the 10mL tube because with one it is too easy to put your hand over the opening to the dounce
  • Used an SPP with a bulb to resuspend the egg pellet
  • Transferred the liquid in the 10mL tube to the dounce with the SPP
  • Unwrapped the pestle and put in the dounce
  • Set up two new 25cm2 flasks
    • Myd88 P1 primary 10/8 MES
  • Added 8mL of medium with serum to one of the new flasks
  • Homogenized for ~30 seconds
    • Pushed down on pestle hard and twisted back and forth briefly, pulled the pestle up, then back down again and twisted again. Repeated 2 more times
  • Used an SPP and bulb to transfer the homogenized liquid to the flask with medium in it
  • Used 5mL serological pipette to mix the medium in the flask 1 time
  • Transferred 5mL of the liquid in the flask to the other flask (identical volumes in both flasks)
  • Laid flasks flat and placed in the 23 degree C incubator
  • Rinsed out:
    • Erlynmyer flask from filtering
    • Dounce homogenizer and pestle
    • Beaker with ethanol
    • Siliconized 10mL glass tube
  • Rinsed with tap water and DI, rinsed the glass tube many times to remove stuck eggs
  • Those were all autoclaved in program 3
  • All pasteur pipettes in the bucket in the TC hood were thrown in the glass disposal box
  • Ethanol wiped the hood
  • Rinsed the tip bucket in the hood, ethanol spray and dried