Cell Culture Preparation of 14091 Myd88 Eggs

2021213 Making Fresh Yeast Paste

  • I made fresh yeast paste by using ~20mL of dry yeast, ~20mL of molecular grade water, and a few mL of apple juice. I added in some extra yeast to get the consistency right. Wanted it to be kind of like royal icing
  • Autoclaved on program 3 twice on 20211213

2021124 Making Yeast Plates

  • Made sure the scooper/spatula was autoclaved
  • In the afternoon, made 2 apple juice yeast plates with 2 streaks of yeast on each plate

20211215 Setting up 14091 Myd88 Flies to Lay

  • Set up 5 vials of 14091 Myd88 flies to lay on the yeast plate at about 2pm

20211216 Day 1 Cell Culture

  • Collected the apple juice plate at ~9:45am and replaced the cage with a new plate for more laying
  • Followed the Prepping Cell Culture Protocol
  • Used freshly made that day 20% FBS Schneider’s medium, and 0% FBS Schneider’s medium:
    • 2 50mL conicals No FBS:
    • 49.5mL Schneider’s Drosophila medium
    • 500ul 100X antibiotics
    • 50ul gentamicin
    • 1 50mL conical 20% FBS:
    • 39.5mL Schneider’s Dosophila medium
    • 10mL FBS
    • 500ul 100X antibiotics
    • 50mL gentamicin
  • There looked like there were some eggs on the plate
  • Used a 100um filter to strain out the eggs, this seemed to keep a lot of yeast debris (brown specks in the image)
  • When looking in the vial, it’s hard to tell how much is debris and what is eggs because the 50% bleach solution turns everything white
  • The cell pellet ended up being pretty small
  • Flasks were left to settle overnight and imaged the next day

20211217 Day 2 Cell Culture

  • Collected the apple juice plate at ~9:45am and disposed of the flies
  • Followed the Prepping Cell Culture Protocol
  • Used the 0% FBS and 20% FBS Schneider’s medium made on the 16th
  • There were many more eggs on the plate this day
  • I used the 70um filter because I wanted to avoid the amount of debris I got yesterday, this should still catch the eggs. There was a huge amount of eggs filtered:
  • The tube/vial seemed to mostly be eggs and not debris
  • Which of course made a large egg pellet, this is the largest egg pellet I’ve ever gotten
  • Flasks were left to settle for a few hours and then imaged:
  • Flasks from 20211216 had very few cells and a lot of debris
  • Flasks from 20211217 had very many cells

Fluid Additions and Imaging

  • 1mL of 20% Schneider’s medium was added to 1 flask from 20211217. These are marked with FA
  • On 20220111 an FA flask and a non FA flask were imaged. They looked similar. There is a lot of debris, most likely junk from dying cells. These cells were pelleted at an incorrect centrifugation speed so this may be why they are unhappy/dying. However it does show that there are some cells that look alive still in the flasks
  • Non FA flask:
  • FA flask: