Fluid Changes, Passing Cells, and More De-Clumping of DinnDiNV 3.0 and 3.1 Cells into Plates

All work was done in the cell culture hood using sterile technique. Most of the Lettered flasks from the last cell work had no growth of cells ad looked mostly dead. All poor looking flasks were thrown out, and any that looked ok were kept and fluid changed (see below).

Fluid Changes

  • ~5mL of fluid was removed from D. innubila primary flasks 1, 1.1, 1.2, and 1.3
  • 5mL of 20% FBS Schneider’s medium with 4% mushroom extract was added back to each flask
  • Additionally, ~5mL of fluid was removed from DinnDiNV 4, 4.1, 4.2, and 4,3 flasks
  • 5mL of 20% FBS Schneider’s medium with 4% mushroom extract was added back to each 4 flask
  • ~3mL of fluid was remoced from D. innubila primary flasks E, C, G.1, and D
  • 5mL of 20% FBS Schneider’s medium with 4% mushroom extract was added back to each flask
  • Additionally, ~5mL of fluid was removed from DinnDiNV 3.0 and 3.1 flasks
  • 5mL of 20% FBS Schneider’s medium with 4% mushroom extract was added back to each 3 flask

Scraping 1 flask

  • The G flask had some areas of growing cells, so I decided to scrape it and try passing it to a new flask
  • Cell scraped the whole flask, and transferred 3mL to a new flask
  • Re-fed the old flask with 5mL of 20% FBS Schneider’s medium with 4% mushroom extract
  • Gave the new flask 5mL of 20% FBS Schneider’s medium with 4% mushroom extract

Dv-1 and S2 cells were passed normally, I will wait until DinnDiNV cells have grown up in the plate before plating S2 with them.

De-clumping “3” cells and plating in a 12 well dish

3.1 trypsinized flow through flask

  • Poured off supernatant
  • Added 5mL trypsin, then immediately poured it off
  • Added 5mL trypsin, and waited 7 minutes until the cells released from the flask surface
  • Placed fluid in a 15mL conical, let settle for 1 minute, then took 4mL of fluid of the top fluid off into a new 15mL conical (presumably holding the single cells/small clumps)
  • Centrifuged both conicals 5 min 200rpm
  • Removed trypsin
  • Resuspended the “top” fluid in 6mL of 20% FBS Schneider’s medium with 4% mushroom
  • Ran this fluid through a 70um cell strainer and used the flow through
  • Plated 1.5mL of the “top” fluid into wells A4, B4, and C4 of a 12 well plate
  • The settled cell fluid pellet was resuspended in 5mL 20% FBS Schneider’s medium with 4% mushroom and plated in a new flask

3.0 cell scraped flow through flask

  • Scrapped whole flask and put fluid in a 15mL conical
  • Let the fluid settle for 1 minute, then took 4mL of fluid of the top fluid off into a new 15mL conical (presumably holding the single cells/small clumps)
  • Centrifuged both conicals 5 min 200rpm
  • Resuspended both conicals in 6mL 20% FBS Schneider’s medium with 4% mushroom
  • Ran both fluids through a 70um cell strainer and used the flow through
  • Plated 1.5mL of the “top” fluid into wells A2, B2, and C2 of a 12 well plate
  • Plated 1.5mL of the “settled” fluid into wells A3, B3, and C3 of a 12 well plate

Placed the plate in the 23C incubator to grow for a few days before using for a transfection experiment