Cell Culture Week of 20220404

Primary Cell Culture Generation and Cell Culture Maintenance Week of 20220404

20220404

• Generated primary cells from myd88 and D. innubila using the updated primary cell culture protocol
  • Made 1 flask of D. innubila (very few eggs, only 1/4 plates had more than ~4 eggs on it)
  • Made 3 flasks of myd88: 1 top and 2 bottom
• Made fresh 20% FBS medium using the 75%:25% old to new FBS
• Using the 75:25 medium, did a 3mL fluid change on:
  • 2 flasks from 02/24 innubila
  • 2 flasks from 02/23 innubila
  • 1 flask from 01/13 innubila
• I had to add some 20% new FBS medium to the 75:25 tube because I did not have enough to do the secondary transfer for the 01/27 flask. These cells might not do as well because of it
• Trypsinized and cell scrapped the 01/27 flask and separated it into 2 new flasks because there was a large build up of precipitate in the flask
  • Poured off medium from flask
  • Added 3mL room temp trypsin and distributed
  • Poured off trypsin
  • Added another 3mL trypsin and distributed
  • Let flask sit for 2 minutes
  • Added 5mL 20% FBS medium
  • Pipetted a few times to get the cells off the side of the flask
  • I noticed they weren’t really coming off, so I used a cell scrapper on them as well to removed the stuck cells
  • Transferred the liquid to a 10mL glass tube
  • Centrifuged tube for 5 minutes at 200rpm
  • Aspirated out the supernatant
  • Resuspended the pellet in 5mL 20% FBS medium
  • Made a new flask with 5mL 20% FBS medium
  • Added the liquid from the tube to the 5mL flask and pipette mixed
  • Separated that flask out into 2, so there were two flasks with 5mL each in them

  20220405

  • Generated primary cells from myd88 and D. innubila using the updated primary cell culture protocol
    • Made 2 flasks of innubila cells, 1 top and 1 bottom
    • Made 3 flasks of myd88: 1 top and 2 bottom