Q：Unusual pH in medium.

A：

· Possible Cause: Incorrect set up for carbon dioxide (CO2) pressure. Suggested Solution: Increase or decrease CO2 pressure in the incubator based on concentration of sodium bicarbonate in medium.

· Possible Cause: No carbon dioxide (CO2) exists in the incubator. Suggested Solution: Monitor carbon dioxide (CO2) pressure and change CO2 tank timely.

· Possible Cause: Overly tight caps on cell culture flasks. Suggested Solution: Loosen caps one-quarter turn or use culture flask with vented cap.

· Possible Cause: Bacterial or fungal contamination. Suggested Solution: Discard cells and the medium.

· Possible Cause: Incorrect balanced salts in medium. Suggested Solution: Use Earle’s salts-based medium in CO2 environment and Hanks’ salts-based medium in atmospheric conditions.

· Possible Cause: Bicarbonate buffer system provides insufficient buffering. Suggested Solution: Add HEPES buffer to attain a final concentration of 10 to 25 mM.

A：

· Possible Cause: Change happens in medium or serum. Suggested Solution: Compare glucose, amino acids, and other components in different media; Increase cell inoculation density; Transfer cells to fresh medium.

· Possible Cause: Lack, depletion or degradation of components essential for cell growth (such as L-Glutamine and growth factors). Suggested Solution: Use fresh medium or add essential components to the medium.

· Possible Cause: The initial cell inoculation density is too low. Suggested Solution: Increase cell inoculation density.

· Possible Cause: Mycoplasma contamination. Suggested Solution: If the culture has been detected to have mycoplasma contamination, discard the culture or use mycoplasma elimination reagent.

· Possible Cause: Cells have been passaged too many times. Suggested Solution: Use new cells.

· Possible Cause: Low levels of bacterial or fungal contamination. Suggested Solution: Use antibiotic medium; If the culture is contaminated, discard it.

· Possible Cause: Medium or serum is stored incorrectly. Suggested Solution: Serum should be stored at -5 to -20 ℃. Medium should be stored at 2-8 ℃ in dark. Complete Medium should be stored at 2-8 ℃ in dark and used within 2 weeks.

A：

· Possible Cause: Cells are overly trypsinized. Suggested Solution: Reduce trypsinization time or trypsin concentration.

· Possible Cause: Poor cell state. Suggested Solution: Use new cells.

· Possible Cause: Mycoplasma contamination. Suggested Solution: Discard the culture if it has been detected to have mycoplasma contamination, or use mycoplasma elimination reagent.

· Possible Cause: No attachment factors in medium. Suggested Solution: For serum-free culture, be sure attachment factors are contained or use coated culture vessels.

· Possible Cause: No carbon dioxide (CO2) exists in the incubator. Suggested Solution: Monitor carbon dioxide (CO2) pressure and change the CO2 tank timely.

· Possible Cause: Temperature fluctuation in the incubator. Suggested Solution: Monitor temperature in the incubator.

· Possible Cause: Too high toxicity of transfection reagents; Too high passage number of cells; Low purity of plasmids. Suggested Solution: Use transfection reagents of low toxicity or change medium timely after transfection; Use cells of low passage number; Use plasmids of high quality.

· Possible Cause: Big cell damage during thawing or cryopreservation. Suggested Solution: Use new cells.

· Possible Cause: Incorrect osmotic pressure of medium. Suggested Solution: Check the osmotic pressure of complete medium. Most mammalian cells can tolerate osmotic pressure of 260-350 mOsmol/kg and most insect cells can tolerate osmotic pressure of 340-380 mOsmol/kg.

·Possible Cause: Excessive toxic metabolites accumulate in the medium. Suggested Solution: Change fresh medium.