(Protocol from Arman Adel,Dec 2nd, 2019)

• 1x PBS
• 0.05% w/v Trypsin (0.53 mmol/l EDTA-4NA with Phenol Red)
• DMEM High Glucose (10% FBS, 1% Pen-strep)

• Remove DMEM from 10cm cell culture dish
• Wash cells with 5ml of 1x PBS (add PBS down side of plate to avoid disrupting the cells)
• Remove PBS from cells
• Repeat step 2-3 (this is not really necessary for HEK293 cells but good for more adherent cell lines)
• Add 1ml of Trypsin to cells (this can be directly applied)
• Place plate in 37˚C cell culture incubator for 2-5 minutes
• Remove plate from incubator and add 5ml to DMEM to trypsinized cells, mix up and down to remove clumps, then add to a 15ml falcon tube
• Optional: Measure cells/ml using bio-rad cell counter (Add 10µl of cells to 10µl Trypan Blue then load 10µl mix onto slide and insert into machine). Look up ‘thermofisher useful numbers for cell culture’ to find specific seeding densities.
• If you did step 8, use the cells/ml concentration and 10cm dish seeding density (2.2×10^6 cells) to determine volume you will add to a fresh 10cm dish(es). Otherwise add anywhere between 300-500µl depending on how confluent the cells appeared.
• Add 10ml of DMEM to 10cm dish(es) with passaged cells
• Place dish(es) in 37˚C cell culture incubators

— Dan Yamamoto-Evans 2019/12/02 20:38