Fluid Changes, Passing Cells, and Trying De-Clumping of DinnDiNV 3.0 and 3.1 Cells

All work was done in the cell culture hood using sterile technique

Fluid Changes

  • ~3mL of fluid was removed from D. innubila primary flasks 1, 1.1, 1.2, and 1.3
  • The fluid was placed in 5mL tubes and frozen at -80 in case of using for PCR analysis
  • 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
  • The fluid was placed in 4 5mL tubes to be frozen at -80 incase of use for virions
  • 5mL of 20% FBS Schneider’s medium with 4% mushroom extract was added back to each flask

Scraping 1 flask

  • The 10/4 flask had some areas of growing cells but also some precipitate, so I decided to scrape it and try passing it to a new flask
  • Cell scraped the whole flask, and transferred 2mL to a new flask
  • Re-fed the old flask with 3mL 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
  • The original flask was labeled A and the new flask was labeled A.1

Putting supernatant from flasks into new flasks

  • For a number of flasks, I decided to try removed the supernatant that contained a lot of floating clumps of cells and try plating that in a new flask, in the hopes that some of those clumps would settle and begin to grow. This is basically a last ditch effort for these flasks
  • Each flask and it’s passaging flask was given a letter
  • For each flask, 2mL of medium from the top of the flask was removed and placed in a new flask. The old and the new flasks got replenished to 5mL of fluid with 20% FBS Schneider’s medium with 4% mushroom extract
  • Flasks:
    • 02/01 2 original flasks, now flasks H and H.1, and I and I.1
    • 01/12 original flask, now G and G.1
    • 01/31 original flask, now D and D.1
    • 09/27 original flask, now C and C.1
    • 12/13 original flask, now B and B.1
    • 01/24 original flask, now F and F.1
    • 01/25 original flask, now E and E.1

S2 and Dv-1 cells were also passed on this day (these get passed every week).

Cell De-Clumping Methods

  • The DinnDiNV cells grow relatively well, but they are consistently clumpy. This seems to be an issue for me because clumps of cells autofluoresce under the GFP light. I researched ways to de-clump cells, and based off of the reagents we currently have in the lab, I decided to try a DNase 1 treatment and cell straining. I got the idea from this protocol, which I roughly followed.
  • Kent also gave me two flasks this week of presumed negative for virus cells, 3.0 and 3.1 cells. Because these flasks had a lot more cells than the 4s I am working on building up, I decided to use these flasks for the de-clumping
  • I tried DNase 1 treatment and cell straining on both flasks, either cell scraped or typsinized
  • For the 3.0 flask:
    • Used the cell scraped on the whole flask
    • Transferred the liquid to a 15mL tube
    • Centrifuged for 5 min at 200 rpm
    • Removed supernatant
    • Resuspended the cell pellet in 600ul 20% FBS Schneider’s medium with 4% mushroom extract
    • Transferred 300ul to a new 15mL tube
    • Plated one of the 300ul of cells in a new flask with 10mL 20% FBS Schneider’s medium with 4% mushroom extract
    • The other 300ul of cells got a DNase 1 treatment: 50ul of Qiagen DNase 1, pipette mixed genetly, and incubated at room temp for 15 minutes
      • Qiagen DNase 1 is potentially 0.6mg/mL stock, so to get 100ug/mL in solution, that is 50ul in 300ul total vol
    • After the 15 minute incubation, I added 3mL 20% FBS Schneider’s medium with 4% mushroom extract
    • Centrifuged for 5 min at 200 rpm
    • Removed supernatant
    • Added 5mL 20% FBS Schneider’s medium with 4% mushroom extract
    • Passed the fluid through a 70um cell strainer
    • Put the flow through liquid in a flask with 10mL 20% FBS Schneider’s medium with 4% mushroom extract
    • Resuspended the cells on top of the strainer in 10mL 20% FBS Schneider’s medium with 4% mushroom extract and put that in a flask
    • Added 20% FBS Schneider’s medium with 4% mushroom extract back to the original flask up to 10mL
  • For the 3.1 flask:
    • Removed all the medium from the flask
    • Rinsed the flask with 3mL of trypsin and poured if off
    • Added 5mL trypsin and waited ~10 min until many of the cells came off the flask
    • Transferred the liquid to a 15mL tube
    • Centrifuged for 5 min at 200 rpm
    • Removed supernatant
    • Resuspended the cell pellet in 600ul 20% FBS Schneider’s medium with 4% mushroom extract
    • Transferred 300ul to a new 15mL tube
    • Plated one of the 300ul of cells in a new flask with 10mL 20% FBS Schneider’s medium with 4% mushroom extract
    • The other 300ul of cells got a DNase 1 treatment: 50ul of Qiagen DNase 1, pipette mixed genetly, and incubated at room temp for 15 minutes
      • Qiagen DNase 1 is potentially 0.6mg/mL stock, so to get 100ug/mL in solution, that is 50ul in 300ul total vol
    • After the 15 minute incubation, I added 3mL 20% FBS Schneider’s medium with 4% mushroom extract
    • Centrifuged for 5 min at 200 rpm
    • Removed supernatant
    • Added 5mL 20% FBS Schneider’s medium with 4% mushroom extract
    • Passed the fluid through a 70um cell strainer
    • Put the flow through liquid in a flask with 10mL 20% FBS Schneider’s medium with 4% mushroom extract
    • Resuspended the cells on top of the strainer in 10mL 20% FBS Schneider’s medium with 4% mushroom extract and put that in a flask
    • For the original flask: rinsed it with 3mL 20% FBS Schneider’s medium with 4% mushroom extract, poured it off, and re-added 10mL 20% FBS Schneider’s medium with 4% mushroom extract to keep the flask going
  • All of these flasks I left in the 23C incubator for 2 days before imaging them in bright field, in GFP, and RPF light. Those images can be seen here