Generating Primary Cell Culture and Inventory for Week of 20220411

20220411 Primary Cell Culture for D. falleni and IMD Null

  • Attempted to do primary cell culture for D. falleni, however after ~16 hours of flies on the small cages with apple juice plates with a small amount of yeast, very few eggs were laid. I decided to let the flies lay on the same plates for another day and try then
  • Followed the updated cell culture preparation protocol for a cage set up with IMD null flies
    • Not very many eggs were laid so only 1 flask of IMD null was made
  • Domeless flasks from 03/30 and 03/31 were fluid changed with 3mL removed and 3mL new fluid added. Before removing the fluid I tried “washing” the surface of the flask because these had a lot of debris on them and I wanted to see if I could get it to come off

20220412 Primary Cell Culture for D. falleni and IMD Null

  • There was enough eggs from the D. falleni to make one primary cell culture flask using the updated cell culture preparation protocol
  • Also followed the updated cell culture preparation protocol for IMD null flies with 1 modification:
    • After homogenization, I transferred the homogenate into a 10mL glass tube
    • Added 3mL of 20% FBS Schneider’s medium to the tube
    • Centrifuged tube for 5 minutes at 2 rpm
    • Aspirated off the liquid
    • Resuspended the cell pellet in 5mL 20% FBS Schneider’s medium
    • Added that 5mL to a flask with another 5mL 20% FBS Schneider’s medium
    • Pipetted to mix that flask
    • Separated out that flask into another flask, so they both have 5mL
  • This was to try to get rid of any debris that would not centrifuge down with such a low centrifugation rate

20220415 Cell Culture Inventory

D. falleni

04/12 1 flask

  • Pretty much nothing in this flask, not cells are visible. I had not gotten very many eggs from these files

D. innubila

04/04 1 flask

  • Not looking great, but there are some good cells
  • Needs fluid change

04/05 1 top and bottom flask

  • Top: lots of debris, but maybe some good cells. Needs fluid change
  • Bottom: also a fair amount of debris, but there are clumps that are still contracting, and some areas with a little grow out of cells. Needs fluid change

04/04 Secondary from 01/27 2 flasks

  • Not a lot in these flasks
  • Some clumps are starting to grow out though
  • Want to leave these alone for a while, might need fluid change soon though

01/13 1 flask

  • There is precipitate in this flask, but there are cells that look like they are growing well
  • Leave these flasks for a while, monitor when to fluid change

02/24 2 flasks

  • Precipitate in this flask
  • Lots of those large dark floating cells, but also some areas where there are long stringy cells growing out and some areas with round cells growing out

02/25 2 flasks

  • Same as the 02/24 flasks - looking promising

Melanogaster

IMD Null
04/11 1 flask

  • Early to tell, but lots of cell clumps that are floating and not attached
  • A good number of cells

04/12 2 flasks

  • Looks like there might be less debris in these flasks
  • Lots of single cells and some cell clumps
  • The large pieces of debris seem to have stayed, but the smaller debris might have been removed

03/23 1 top and bottom flask

  • Top: A number of cells and clumps that look alive, but not much evidence of growth. Kept flask
  • Bottom: Same as top flask

03/24 1 top and bottom flask

  • Top: lots of debris, seems very unlikely for the cells to grow out, thrown out
  • Bottom: same as top flaks

Domeless
03/30 top and bottom 2 flasks

  • Top: very covered in debris: thrown out
  • Bottom: flask has a lot of debris, but there are some alive cells, keep flask for now

03/31 top and bottom 3 flasks

  • Top: very covered in debris: thrown out
  • Bottom 1 and 2: lots of debris, almost no alive looking cells left. Not a lot of floating debris, but the debris on the flask are all still here. Looks like “washing” the flask did not work

03/09 1 bottom flask

  • A little bit of evidence of the thin stringy cells growing, just very slowly

SPZ
03/03 top and bottom 2 flasks

  • Top: some cells but there is so much debris in the flask: thrown out
  • Bottom: clumps of cells still contracting, lots of evidence of growth. Might want to try transferring this one, there is a fair amount of debris as well

Myd88
04/04 top and bottom 3 flasks

  • Lots of debris in all flasks and no evidence of cells growing out. All flasks were thrown out

04/05 top and bottom 3 flasks

  • Huge amounts of debris, very little evidence of growth or alive cells. All flasks were thrown out

03/30 Secondary from 02/03 Trypsin 3 flasks

  • Lots of floating clumps of cells
  • Some clumps have attached and started to spread out a little
  • Clumps may have been to big/not enough time on the trypsin

03/30 Secondary from 02/03 Cell Scraper 3 flasks

  • Lots of individual cells
  • Floating cells and clumps of cells
  • Many cell “types”

02/03 1 flask

  • Looking very good still
  • Some areas look almost “confluent”
  • I am thinking about spreading this out over 4 flasks with a cell scraper and doing a centrifuge in between