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Subculture (Old)

Materials:

• ATV* (TRYPSIN) solution:
• 0.05% Trypsin-0.5 mM EDTA-137 mM NaCl-5.4 mM KCl (0.058% NaHCO₃)
• Medium (5% FBS)
• PBS or Hanks

*See protocol recipe.

Protocol:

1. Withdraw medium, rinse with PBS or HANKS, aspirate and add ATV (37° C, e.g. 2 ml/T25 flask, 3 ml/10 cm tissue culture dish), roll back and forth over cells for 1 to 2 minutes.
2. If cells are not coming off rounding up for transfer within 5 minutes, place dish (or flask) at 37° C. Replace trypsin stock if cells do not quickly respond to trypsin.
3. Tap on both sides of flask or dish to release cells. Using a small pipette, gently spray off the cells from the plastic. (It is important to attain as many single cells as possible at this point).
4. Add appropriate amount of medium (bring total volume to 10 ml) and pipette cell suspension up and down a few times.
5. Centrifuge at 1000 rpm (1600 g) for 5 minutes in a 15 ml conical tube. Aspirate off supernatant, and immediately resuspend in 10 ml of fresh medium.
6. Count the cells with a hemacytometer and sow cells for propagation dishes: plate 1x10⁵ cells and 2x10⁵ per 10 cm TCD. It is very important to have the majority of the cells as single cells, to have the cells spread evenly in sowing, and for the sowing density to be within the correct range.
7. Cells used for either biochemical or morphological experiments are plated at 5x10⁵ cells per 10 cm TCD. For morphology 6 glass 1x1 cm cover slips are placed in the bottom of a 10 cm TCD. The next day, after cells are attached, cover slips are removed and placed in individual 6 cm Falcon dishes. Use 4 ml of medium for 6 cm dishes.