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Converting Adherent Cells to Suspension Cells (Serum Dilution)

Protocol

  1. Begin with cultures at maximum cell density.
  2. Dissociate the cell monolayer using standard procedures. Centrifuge and resuspend the cell suspension with serum-free medium containing 5% serum (V/V).
  3. Count the cell suspension, and then seed two or more flasks with 3-5*10^5 viable cells/mL in serum-free medium containing 5% serum (V/V). The density may need to be adjusted for different cell lines.
  4. Observe the cultures daily. Remove samples and record the number of viable cells for each flask.
  5. Incubate cells in standard condition until the maximum cell density is achieved.
  6. If the cell viability is >85% and the generation time is similar to that observed with original medium, it suggests that the cells adapt to growth in current condition. Then repeat steps 2-5 using a lower concentration of serum at each split. The recommend beginning is at 5% serum and then lower to 2%, 1%, 0.5% and finally 0.1% prior to eliminating serum from the culture.
  7. If the cell viability is<80% and the generation time is significantly increases in the serum condition, increase the serum level to the previous value until the cells adapt to growth again (Two split cycles at least are recommended). Then repeat steps 2-5 using a lower concentration of serum at each split.

Note

1) Not all cell lines can be adapted to suspension growth.
2) It takes a long time to convert adherent cells to suspension cells.
3) The cells, which is successfully converted, may differ with the original cell population in some characteristics.
4) Some cells may require a small amount of serum for growth. Adding 0.1%-0.5% serum for culture is acceptable.

Cell culture condition

  • Mammalian cells: 95% Air, 5% CO2, 35°C-37°C
  • Invertebrate cells: Air, 25°C-27°C
For research use only. Not intended for any clinical use.

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