Gibco™ Sf-900™ II SFM

Cells can be adapted by Sequential or Direct Adaptation. Suggested protocols for each are below, and you can also find more information by searching "Adaptation of Cell Cultures to a Serum-Free Medium" from our website home page.

SEQUENTIAL ADAPTATION
1) Subculture the cells growing in serum-supplemented medium into a 25%:75% mixture of SFM and serum supplemented medium.
2) When the cell density is 5 x 10E5 cells/ml, subculture the cells into a 50%:50% mixture of SFM and serum supplemented medium at a cell density 2.5 x 10E5 to 3 x 10E5 cells/ml.
3) Continue to subculture after the cell density 5 x 10E5 cells/ml in gradually increasing proportions of SFM until the serum is ~0.1% with about 85% cell viability.
4) Subculture the cells into SFM with an innoculum of 2.5 x 10E5 to 3 x 10E5 cells/ml.
5) When the cell density is 1 x 10E6 to 3 x 10E6 cells/ml (4 to 6 days post planting) subculture the cells again.
6) Stock cultures of SFM adapted cells should be subcultured in SFM every 3 to 5 days when the cell density is 1 x 10E6 to 3 x 10E6 cells/ml with 90% viability.

DIRECT ADAPTATION
Some cells can be directly adapted from serum-containing medium to SFM. For direct adaptation, the cell innoculum should be 1.5 x 10E5 to 3 x 10E5 cells/ml.
Cells should be subcultured when the cell density is 1 x 10E6 to 3 x 10E6 cells/ml. Cells are fully adapted to SFM when the cell density is 2 x 10E6 to 4 x 10E6 cells/ml after 4 to 7 days in culture.
Stock cultures of cells adapted to SFM should be subcultured in SFM every 3 to 5 days when the cell density is 1 x 10E6 to 3 x 10E6 cells/ml with 90% viability.

Some cells, such as insect cells, are sensitive to changes in their medium. By sequentially adapting cells, the medium is changed with minimal effects on cell growth.

It should be okay to thaw the cells into Sf-900 II SFM. This is a richer media compared to the Sf-900 III SFM so the cells would have an easy time adapting. We would recommend taking the cells through 3 passages in the new medium before using them for any experiments as that they have enough time to adapt.

Many antibiotics are suitable for use with insect cells. The following antibiotics are commonly used:
- Penicillin/Streptomycine: 50-100 U/mL; 50-100 µg/mL
- Amphotericin B (Fungizone antimycotic): 0.25 µg/mL
- Gentamicin: 0.5 mL of 10 mg/mL solution in 500 mL media (final concentration: 10 µg/mL)

Heat inactivation is not necessary. Our team has routinely used serum that has not been heat-inactivated, and we have not observed any effect on cell growth or morphology.

Many cells do not require heat-inactivated FBS. Some cells prefer heat-inactivated FBS. For instance, we use heat-inactivated FBS for our insect cell lines, i.e., Sf9 and Sf21 cells.