Myd88 Egg Collection and Cell Culture Prep with Kent (watching) 2021 10 07

Egg Collection

  • Brought the second plate to the fly room
  • Set up the CO2 plate at the station (turned on CO2, plugged in hose and turned valve)
  • Flipped over fly cage onto the CO2 plate (top of the cage is mesh)
  • Tapped down until all the flies are on the bottom and sleeping
  • Took off the red end and squeezed to keep the plate in place
  • Added in the second plate, and put the red part back onto the cage
  • Let the flies wake up horizontally for a few minutes, then turned it upright and left for the next day
  • We looked at the plate under the microscope: looks of effs on the plate, some visible in the agar, probably some in the yeast but we can’t see them. There are a few - hatched eggs already, we don’t want those though
  • Myd88 might have difference hatching time than other flies, they were set up at 2pm ish yesterday, collected at ~9am

Preparing Solutions for Filtering

  • New 70% ethanol
    • Rinsed graduated cylinder
    • Ethanol 200 proof in the flammable cabinet
    • 700mL ethanol in the cylinder
    • Added distilled water to 1000mL
    • Poured into a glass bottle on Kent’s bench for ethanol
    • Rinsed cylinder with tap water then distilled water
    • Poured out 70% ethanol into a small beaker on the bench
  • Bought from the store: 50mL tubes, a large and small squirt bottle, and 2 containers of molecular grade water
  • New 1X wash
    • 100mL 10X wash (used 50mL serological pipette)
    • 900mL molecular grade water
  • Added molecular grade water to the large squirt bottle
  • Added 1X wash to the small squirt bottle
  • Made 50% bleach solution (for de-chorionating the embryos and sterilizing)
    • In a 50mL conical
    • 10mL bleach (under sink)
    • 10mL 1X wash

Filtering

  • Used tweezer to pick off dead flies from the yeast and into the ethanol beaker
  • Set up filter
    • Autoclaved flask at the bottom, yellow filter(100um, then green filter (400um), then funnel
  • Took the egg plate and squirted a layer of water on it
  • Used a paint brush to mix up the whole plate
    • Don’t dig into the agar
    • Want to break up the yeast/make it homogenous
  • Tipped the plate over into the funnel
  • Squirted water to rinse all the fluid into the funnel
  • Repeated adding water to the plate because there were some yeast chunks left, brushed, and washed again
  • Rinsed brush when done in the ethanol beaker
  • Took off the funnel and the green filter
  • Rinsed yellow filter with water
  • Rinsed the yellow filter with 1X wash
  • Squirt around the edges of the filter
  • Wash for maybe 2-ish minutes, hard to know if the yeast is out but want to rinse for a while
  • Got a new 50mL conical
  • Flipped over the yellow filter into the conical opening
  • Pipetted 10mL 50% bleach/wash solution slowly into the yellow filter to release the eggs
  • Pipetted out the 10mL immediately (so the eggs don’t stick) into a glass 10mL siliconized tube
  • Let the tube sit upright for 2-3 min to soak (pretty quick)
    • The eggs that are fertilized should start to settle out
  • After the few min, centrifuge to pellet the embryos: program 2 (400rpm 3 min)

In Tissue Culture Hood

  • Used siliconized pasteur pipette to aspirate off the bleach liquid (using vac)
  • Added 10mL 420 medium without serum to the glass tube (medium made on 20211005)
  • Inverted glass tube to mix and centrifuged in program 2
  • Washing off the bleach
  • After centrifugation, not all the eggs had pelleted but most of them had
  • Aspirated off liquid from the center of the tube with a new pasteur pipette and the vacuum
  • Added another 10mL of media without serum and inverted to mix
  • Centrifuged in program 2
  • Aspirated off liquid from the center of the tube with a new pasteur pipette and the vacuum
  • Added another 10mL of media without serum for a total of 3 washes
  • Used a siliconized pasteur pipette and a bulb to try to pipette disrupt the eggs on the side of the tube (didn’t work to well)
  • Centrifuged in program 2
  • Set the vacuum pretty low
  • Used siliconized pasteur pipette and vac to aspirate until 200-300ul left
  • Added 1.8mL of media with serum to the glass 10mL tube (2mL total)
  • Made 2 25cm2 flasks with label: myd88 P1 primary 10/7
  • Added 8mL of media with serum to one of the 25cm2 flasks
  • Unwrapped the glass dounce (autoclaved) and set the receptacle in the 15mL rack in the TC hood
  • Used a fresh siliconized pasteur pipette and bulb to resuspend the pelleted cells and transfer the liquid to the dounce receptacle
  • Unwrap and put in the pestle
  • Pushed down and twisted firmly on the pestle: but only for ~10 seconds! Pretty quickly, don’t want to over-mash
  • Sucked up the liquid in the receptacle with a new siliconized pasteur pipette and bulb and added it to the 25cm2 flask with medium in it already
  • Remove 5mL of the liquid from the first flask and add it to the unused flask
  • Lay the flasks flat
  • Looked at them in the scope immediately
    • Looks like single cells and good clumps of cells
    • Also looks like there is less yeast debris
  • Put the flasks in the 23 degree incubator

Cleanup (w/out Kent)

  • Rinsed out:
    • Erlynmyer flask from filtering
    • Dounce homogenizer and pestle
    • Beaker with ethanol
    • Siliconized 10mL glass tube
  • Rinsed with tap water and DI
  • The 10mL tube had a lot of stuck eggs on it
  • I rinsed many many times, shook the tube with water in it and vortexed it for a long long time to remove as many eggs as possible
  • Items above were prepared for autoclaving (foil wrap or lid, autoclave tape) and put in the autoclave under 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
  • Closed the hood sash, turned off the blower and the light