Cell Culture Inventory And Plating 3.1 Cells for Transfection on 5-23

Cell Culture Inventory

3/22 ovaries

  • 1 flask
  • Flask full of debris
  • I don’t see any alive cells
  • Flask was thrown out

4/3 and 4/11 primaries

  • 2 flasks for each date
  • Supernatant flask is very full of debris
  • Other flask is mostly full of dead cells, doesn’t seem like anything is alive in either flask
  • Flasks were thrown out

1.1 innubila P2

  • 1 flask
  • A couple of good areas of growing cells
  • Want to fluid change
  • This flask may have enough cells to scrape and try re-plating again

1.2 innubila P2

  • 1 flask
  • Some small clumps of cells growing ok
  • Want to fluid change

1.2.1 innubila P3

  • 1 flask
  • Some small clumps of cells growing ok
  • Want to fluid change

1.3 innubila P2

  • 1 flask
  • Has a good number of good looking areas of cells, but this flask also has more precipitate than other flasks
  • Want to fluid change

1.3.1 innubila P3

  • 1 flask
  • Some small clumps of cells growing ok
  • Want to fluid change

1.4 innubila P2

  • 1 flask
  • Some small clumps of cells growing ok
  • Want to fluid change

1.5 innubila P2

  • 1 flask
  • Only one small area of growing out cells…
  • Want to fluid change

1 innubila P1 from 1/12

  • 1 flask
  • Basically this flask is entirely full of precipitate
  • Flask was thrown out

G and C innubila primaries

  • 3 flasks
  • All are very full of precipitate
  • Flasks were thrown out

E, D, and G.1 innubila Primaries

  • 3 flasks
  • Many dead cells and precipitate with nothing growning
  • Flasks were thrown out

Passing 14091 Myd88 cells

  • Cell scraped p26 flask
  • Used 1mL to seed 2 new flasks
  • Each flask was suspended with 5mL of 10% FBS Schnedier’s medium

Fluid Exchanges

  • Removed 3mL from each flask
  • Added back in 3mL of 20% FBS, 4% mushroom broth Schneider’s medium
  • Flasks:
    • 1.1
    • 1.2
    • 1.2.1
    • 1.3
    • 1.3.1
    • 1.4
    • 1.5

Plating 3.1 Cells in Plate 9

  • Wanting to do another transfection experiment, so I want to use the 3.1 cells in a 12 well plate
  • Took the 3.1 P3 flask and poured of the medium
  • Added 5mL trypsin and immediately poured off
  • Added 5mL trypsin and waited ~5-10 minutes for the cells to release from the bottom of the flask
  • Sucked up the liquid and put it into a 15mL tube
  • Centrifuged the tube for 5 min at 200rpm
  • Removed the trypsin
  • Resuspened the cells in 10mL of 10% FBS, 4% mushroom Schneider’s medium
  • Ran the cell fluid through a 70um filter
  • Resupended the cells stuck on the filter in 10mL 10% FBS, 4% mushroom Schneider’s medium and added that to 2 new flasks for a passage 4
  • For the 10mL fluid that went through the strainer, 1.5mL of this was added to wells A2, B2, C2, A3, B3, and C3 of plate 9 (see layout)
  1   2   3   4  
A S2 control   3.1 DinnDiNV control, 100ul schneider’s medium   3.1 DinnDiNV control, 100ul schneider’s medium   X  
                 
                 
B S2 2020 1ul reagent   3.1 DinnDiNV, 1ul 2020 reagent, 1ul pAc5   3.1 DinnDiNV, 1ul 2020 reagent, 1ul pAc5   X  
                 
                 
C S2 2020 1ul reagent   3.1 DinnDiNV, 1ul 2020 reagent, 1ul pAc5   3.1 DinnDiNV, 1ul 2020 reagent, 1ul pAc5   X  
                 
                 

Passing 4.3 DinnDiNV cells

  • I make sure to finish all of my other cell work before starting on these cells because they are persistently infected with DiNV
  • Going to use trypsin to pass these cells
  • Poured off the medium
  • Added 5mL trypsin then poured it off
  • Added another 5mL trypsin and waited ~5-10 min for the cells to release from the flask
  • Added the fluid to a 15mL tube and centrifuged for 5 min at 200rpm
  • Removed the supernatant from the cell pellet
  • Resuspended the cells in 12mL 20% FBS, 4% mushroom Schneider’s medium
  • Separated out that fluid into 2 new flasks for passage 2