Passing Cells and Checking on Plated Cells 05/03

Plated 3.0 and 3.1 cells

I wanted to check on the 3.0 and 3.1 cells that I plated on 4/27 to see if they had grown up enough to try plating S2 cells into the plate. It has been 6 days after they were plated, but it turns out they haven’t grown very much. However they were very sparse 1 day after plating.
Well A2 (scraped cells) on 4/28: Well A4 (tyrpsined cells) on 4/28:

The cells have definitely grown since then, but not enough to be ready for them to be transfected soon:
Well A2 (scraped cells) on 5/3: Well A4 (trypsined cells) on 5/3: Well B4 (trypsined cells) on 5/3:

I think I will wait until Sunday maybe to plate the S2 cells. For each well in the 12 well plate with cells I added 1mL of 20% FBS Schneider’s medium with 4% mushroom extract.

1.2 and 1.3 cells

These flasks are the best looking of my primary innubila originally from 01/12. Because I think continually passing these cells is what might be keeping them going, I am going to scrape these two flasks and pass them again. This is what 1.3 looks like on 5/3: Note that these nice clumps of cells are really only in the middle of the flask, it’s not near confluent.

For each of these, I scraped the in the center of the flask where the most groups of cells are, then transferred 3mL into a new flask. Then each flask got 5mL of fresh 20% FBS Schnieder’s medium with 4% mushroom extract.

Passing Cells

S2 and Dv-1 cells were passed into new flasks with 100ul of cell fluid and 5mL of 10% FBS Schenider’s medium in each flask.

Myd88 cells were passed with trypsin. Fluid was poured off of the flask, then 3mL trypsin was added and washed around. Then the trypsin was poured off. Then another 5mL of trypsin was added and incubated on the flask for ~5min. Not all the cells came off the flask at that point, but a good number did. The fluid was removed from the flask and centrifuged 5 min at 200rpm. The supernatant was removed, and the cells were resuspended in 2mL 10% FBS Schenider’s medium. 1ml was used to reseed the new flasks, which were filled with 5mL 10% FBS Schenider’s medium each.