2. Wash cells with PBS or HBSS (see Table 1), aspirate, and discard. Repeat.

Tip - The volume of PBS or HBSS should be approximately the same as the volume of medium used for culturing the cells.

3. Add enough warmed 1x trypsinEDTA solution (see Table 1) to cover the monolayer, and rock the flask/dish 45 times to coat the monolayer.

4. Place the flask/dish in a CO2 incubator at 37C for 12 min.

5. Remove flask/dish from incubator and firmly tap the side of the flask/dish with palm of hand to assist detachment.

Tip - If cells have not dislodged, return the flask/dish to the incubator for a few more minutes.

IMPORTANT: Do not leave cells in 1x trypsinEDTA solution for extended periods of time. Do not force the cells to detach before they are ready to do so, or clumping may occur.

Tip - Overly confluent cultures, senescent cells, and some cell lines may be difficult to trypsinize. While increasing the time of trypsin exposure may help to dislodge resistant cells, some cell types are very sensitive to trypsin and extended exposure may result in cell death. In addition, some cell lines will resist this treatment and will produce cell clumps.

6. Once dislodged, resuspend the cells in growth medium containing serum.

Tip - Use medium containing the same percentage of serum as used for growing the cells. The serum inactivates trypsin activity.

7. Gently pipet the cells up and down in a syringe with a needle attached to disrupt cell clumps.

Tip - If pipetted too vigorously, the cells will become damaged. Ensure
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