Optimally 3 or 4 d after picking colonies into the 96-well plates, the cells are at a density required for splitting. Since cells in different wells generally exhibit different growth potential, they will not grow at a synchronous rate. Therefore the optimal time for splitting the whole plate needs to be determined. It is best to choose a time when the majority of the cells have reached 80-90% confluency. Another more laborious alternative is to passage the clones at different stages (pooling them into groups depending on their growth rate) and replating them into different 96-well plates.
1. To passage cells in 96-well plates, first prepare several gelatinized 96-well plates.
2. Add 200 pL medium/well, and place plate in a 37°C incubator.
3. Aspirate the medium from the plate to be split, and then wash with PBS by multipipeting 200 pL of PBS into each well followed by aspiration.
4. Remove all traces of PBS, and then add 50 pL trypsin per well.
5. Incubate at 37°C for 5-10 min. The cells should detached with gentle tapping on the plate.
6. Multipipet 50 pL medium/well into each of the wells. Pipet up and down about five times so as to resuspend completely (see Note 11). Then split them (working row by row) into two or three newly gelatinized plates.
7. Return these plates to the 37°C incubator (see Notes 12 and 13).
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