Note: all reagents should be pre-warmed.
Assuming growth in a T75...
- 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.
- Aspirate culture medium, and rinse cells with a few mL PBS.
- Aspirate PBS, and add 3 mL of 025% Trypsin (with EDTA). Incubate cells until they release from surface - typically 2-3 min.
- Triturate cells with 4 mL medium, and transfer to fresh conical tube.
- For best recovery, rinse empty T75 with another 3 mL of medium, and add to the cells.
- Centrifuge at 500 g for 5 min.
- 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.
- 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).
- Medium should be exchanged every day according to ATCC, but cells do fine with every other day if rapid growth is not a necessity.