20.109-Protocols/Propagating J1

Note: all reagents should be pre-warmed.

Assuming growth in a T75...

1. Estimate how many new culture flasks will be needed, and treat each with 5 mL 0.1% gelatin (in D-PBS) for at least 10 min.
2. Aspirate culture medium, and rinse cells with a few mL PBS.
3. Aspirate PBS, and add 3 mL of 025% Trypsin (with EDTA). Incubate cells until they release from surface - typically 2-3 min.
4. Triturate cells with 4 mL medium, and transfer to fresh conical tube.
5. For best recovery, rinse empty T75 with another 3 mL of medium, and add to the cells.
6. Centrifuge at 500 g for 5 min.
7. Resuspend and count cells to determine plating ratio.
   • Taking 90 μL of cells from a 10 mL suspension and mixing with 10 μL of Trypan tends to work well.
8. Plate 3-4 M cells per flask, depending on desired day of collection.
   • In two days, 3M cells will grow to ~10-20M cells (variety due in part to incubator conditions).
9. Medium should be exchanged every day according to ATCC, but cells do fine with every other day if rapid growth is not a necessity.