Cell Culture Preparation of 33098 Dicer Eggs

20211115 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 20211115

202111116 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

20211117 Setting up 33098 Dicer Flies to Lay

  • Set up 5 vials of 33098 Dicer flies to lay on the yeast plate at about 2pm, this has been the most flies I’ve used so far

20211118 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 0% FBS medium made on 20211109 and 10% FBS medium made on 20211102
  • Used Trypsin opened 20211111
  • For added sterility: after every time doing something out of the TC hood, I sprayed my gloves with ethanol before going back into the hood (centrifuges, etc.)
  • Yield of eggs was very good
  • Flasks were left to settle overnight and looked at the next day
  • Supplies were put in the autoclave in the lab to sterilize for the next day
  • New 0% FBS and 10% FBS 420 mediums were made for the next day

20211119 Day 2 Cell Culture

  • Collected apple juice plate at ~9:30am, flies were placed in the morgue
  • Followed the Prepping Cell Culture Protocol
  • Used 0% FBS made on 20211109 and 20211118, and 10% FBS made on 20211118
  • Opened new Trypsin: 20211119
  • For added sterility: after every time doing something out of the TC hood, I sprayed my gloves with ethanol before going back into the hood (centrifuges, etc.)
  • Again there was a lot of eggs, the most I’ve ever gotten. I think I hit a great amount of files in the cage for laying with five vials
  • Flasks were left to settle over the weekend before looking at them

20211122 Imaging Flasks After the Weekend

Flasks from 20211119: lots of single cells that look really good. At 4X magnification you can see that there’s a lot of them, most of the specs are cells I had a problem again with the cell pellet after homogenization and while washing with medium, it did not want to break up. I think these are clumps of cells re-stuck together after homogenization. I used pipette mixing to break them up a little in the flasks, but they still do show up. Hopefully there is not much “junk” in these, and it’s mostly just cells.

Flasks from 20211118: similar to the 19th, many cells, some clumps. Obviously it’s early, but they all look good.