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.
 * 8) *Taking 90 &mu;L of cells from a 10 mL suspension and mixing with 10 &mu;L of Trypan tends to work well.
 * 9) Plate 3-4 M cells per flask, depending on desired day of collection.
 * 10) *In two days, 3M cells will grow to ~10-20M cells (variety due in part to incubator conditions).
 * 11) Medium should be exchanged every day according to ATCC, but cells do fine with every other day if rapid growth is not a necessity.