Cell Culture Work for the Week of 20220328: Domeless Primary Cell Generation, Fluid Changes, Secondary Transfer, and Inventory

20220330

  • Prepared primary cell culture from Domeless flies using the updated primary cell culture protocol. This generated 1 top flask and 2 bottom flasks
  • Made new 20% serum medium with 75% old lot # FBS 20% new lot # FBS
  • Used the 75:25 medium to do a fluid exchange on:
    • 03/09 domeless bottom 1 flask
    • 03/03 SPZ top and bottom flasks (1 each)
    • 02/23 IMD null (2 flasks)
    • 1 flask from myd88 02/03
  • Used the new lot # 20% FBS to do a fluid exchange on:
    • 03/23 IMD null top and bottom flasks (1 each)
    • 03/24 IMD null top and bottom flasks (1 top, 2 bottom)
  • For fluid change
    • Remove 3mL from the flask
    • Add in 3mL new medium
    • Gently redistribute liquid over the flask to mix
  • Did a secondary transfer on the other 2 02/03 myd88 flasks, one using a cell scraper and one using trypsin. For both of these I used the 75:25 FBS medium
  • Cell scraper:
    • Made a new flask with 10mL of new 20% FBS medium
    • Unwrapped the cell scrapper and used it on the the surface of the flask
    • Gently scrapped sideways across the flask many times until it looked cleared up and there was a lot of visible stuff in the medium
    • Transferred the liquid from the flask into the new flask with the 10mL of medium
    • Pipette mixed the flask
    • Transferred 5ul from that flask into 2 new flasks, making a total of 3 new flasks from the first one
    • Labeled these flasks SC
  • Trypsin
    • Poured off the medium from the flask
    • Added 3mL of room temp trypsin to the flask and distributed it over the surface
    • Immediately poured off the trypsin
    • Added another 3mL of trypsin to the flask and distributed it over the surface
    • Incubated the trypsin on the flask for 2 minutes
    • Added 6mL of 20% FBS medium to the flask
    • Pipetted the liquid a couple of times over the flask surface to release the stuck cells
    • Transferred the liquid to a glass 10mL tube
    • Centrifuged the tube for 5 minutes at 200rpm
    • Gently removed the supernatant (pellet was visible)
    • Resuspended the cell pellet in 5mL of 20% FBS
    • Made a new flask with 10mL of 20% FBS
    • Transferred the liquid in the 10mL flask into the new flask
    • Pipette mixed the flask
    • Separated out that flask into 2 more flasks with 5mL each, producing 3 total flasks
    • Labeled these flasks T

20220331 Cell Culture Inventory

IMD Null

  • 02/23 (2 flasks)
    • There are some cells in this flask that look alive, but there has been no growth really after a month
    • These flasks were thrown out
  • 03/23 top
    • Lots of clumps of cells that look good, but not seeing any evidence of grow out yet
    • These are still “young” so it’s worth waiting on them
  • 03/23 bottom
    • Saw a clump of cells contracting
    • There are a good number of clumps of cells, just none really growing out and spreading yet
  • 03/24 top
    • Lots of debris and not very many cells
    • This flask was thrown out
  • 03/24 bottom (2 flasks)
    • Some debris pile up
    • There are some good clumps but not very many cells in these flasks

Domeless

  • 03/09 top
    • Not a lot of cells and no evidence of the cells growing out
    • This flask was thrown out
  • 03/09 bottom 1
    • Found a clump of cells contracting still
    • Not a lot of cell growth but I’m going to keep with this one
  • 03/09 bottom 2
    • Some clumps of cells but not very many cells, no cells growing out
    • This flask was thrown out
  • 03/30 top
    • Lots of single cells and a fair amount of debris too
  • 03/30 bottom
    • A good number of single cells and clumps, hopefully these flasks will be good
  • 03/31 top
    • Lots of single cells and not a lot of clumps of cells
    • This flask was looked at very recently after generation and there was very little debris (probably not settled yet)
  • 03/31 bottom 1 + 2
    • A lot of single cells and a good amount of cell clumps
    • This flask was looked at very recently after generation and there was very little debris (probably not settled yet)

SPZ

  • 03/03 top
    • Lots of debris but there are some cells and clumps alive. I will keep this one going for a bit
  • 03/03 bottom
    • I saw a cell clump contracting
    • Lots of clumps of cells and evidence of cells growing
    • Might want to think about doing a secondary transfer on this flask soon, but want to keep an eye on for a bit first

Myd88

  • 02/03 original flask (1)
    • Still a good number of cells alive and presumably growing. There are lots of areas of the flask that are covered in a monolayer of cells
  • 03/02 fluid transfer from 02/03 flasks (3 flasks)
    • Lots of the huge dark cells that float, I feel like these cells aren’t alive?
    • There are a few cells that have settled on the bottom
    • Might be a good idea to fluid change this flask
  • 03/30 cell scrape from 02/03 (3 flasks)
    • Hard to tell yet what is going on, they’ve only been in the flask overnight
    • There are a lot of single cells, some big cell clumps, some of the large floating brown cells
    • There were a few spots with a couple (~6) of the smaller round cells that look like they might have divided out
  • 03/30 trypsin from 02/03 (3 flasks)
    • Again, it’s hard to tell how these are doing after 1 day
    • There are more clumps in these flasks, and not very many single cells
    • What’s surprising is that there’s basically no debris! Seems like washing out stuff and centrifuging got rid of it
    • There are some places that look like cells might be growing out, too early to tell

Innubila

Note: innubila flasks had some things done to them by Kent in the last few weeks, I’m not sure exactly what he did with them but I made guesses from the notes on his flasks

  • 03/17 - there should have been 2 flasks but I only have 1
    • Lots of cell debris and hardly any cells at all
    • This flask was thrown out
  • 03/18 - I know I made cells this day but I don’t have a flask anymore. My guess is that Kent threw it out without telling me. Likely it wasn’t a good flask anyways. Or he never gave me back this flask
  • 02/18
    • This was fluid changed by Kent on 3/4 and 3/23
    • There were almost no cells and all debris
    • This flask was thrown out
  • 02/24 (2 flasks)
    • Was fluid changed by me on 03/03
    • Lots of those large dark cells that sort of look dead
    • One of these flasks has a lot of growth of the long skinny cells
    • These flasks need another fluid change
  • 02/25 (2 flasks)
    • Was fluid changed by me on 03/03
    • Lots of the long stringy cells, the big brown cells, and some smaller round cells
    • These flasks need another fluid change
  • 01/13 (1 flask)
    • I’m not exactly sure what happened to this flask. It was fluid changed by me on 02/10, then by Kent on 2/18, 3/2, 3/7, and 3/23
    • It also seems like Kent trypsinized this flask on 3/7, but I don’t know why there are still cells left if that happened?
    • Anyways, there are some single cells and also some areas where there are a lot of those small round cells growing out
    • I should fluid change this flask soon
  • 01/27
    • This flask was fluid changed by me on 2/23 and by Kent on 3/4 and 3/23
    • This flask has a lot of precipitate crystals from the media
    • But there are still cells that might be a love, round ones and the stringy ones
    • I might try transferring these to a new flask to get rid of the crystals?
  • 02/04 (2 flasks)
    • Fluid changed by me on 2/23, Kent on 3/4 and 3/23
    • Lots of debris and floating things, no growing cells
    • These flasks were thrown out