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Culture Medium: Current | Culture Medium: Old
| Culture Conditions | Recipes: Current |
Recipes: Old | Cell Thawing |
Characteristics | Subculture: Current | Subculture: Old |
Quality Control | Cell IMF Protocols
| Preservation (Cell Freezing) | DNA Delivery
- 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.
- 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.
- 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
- 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).
- Add appropriate amount of medium (bring total volume to 10 ml) and pipette
cell suspension up and down a few times.
- 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.
- 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.
- 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.