|Cells were stored incorrectly|
- Obtain new stock and store in liquid nitrogen. Keep the cells in liquid nitrogen until thawing
|Home made freezer stock is not viable|
- Freeze cells at a density recommended by the supplier
- Use low-passage cells to make your own freezer stocks
- Follow procedures for freezing cells exactly as recommended by the supplier. Note the freezing procedure recommended by this handbook is a general procedure provided as a guideline only.
- Obtain new stock
|Cells were thawed incorrectly|
- Follow procedures for thawing cells exactly as recommended by the supplier. Note the thawing procedure recommended by this handbook is a general procedure provided as a guideline only.
- Make sure that you thaw the frozen cells quickly, but dilute them slowly using pre-warmed growth medium before plating.
|Thawing medium is not correct|
- Use the medium recommended but the supplier. Make sure the medium is pre-warmed
|Cells are too dilute|
- Plate thawed cells at high density as recommended by the supplier to optimize recovery.
|Cells not handled gently|
- Freezing and thawing procedures are stressful to most cells. Do not vortex, bang the flasks to dislodge the cells (except when culturing insect cells), or centrifuge the cells at high speeds.
|Glycerol used in the freezing medium was stored in light (if applicable)|
- If stored in light, glycerol is gets converted to acrolein, which toxic to cells. Obtain new stock.