Image showing Sf9 cells in suspension (flask) and in transfection stages (6-well plate). The Sf-900 II media being used here is warmed and supplemented with heat inactivated FBS, antibiotics and L-glutamine.
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The use of insect cells and lytic baculoviruses is now one of the most widely used systems for the production of recombinant mammalian proteins. Many post-translational modifications are made correctly in insect cells and proteins unable to be expressed in E. coli have been successfully expressed in the insect cell system. Expression of the highly abundant baculovirus polyhedrin gene is non-essential for viral replication in tissue culture and consequently, this gene may be deleted and its strong promoter coopted for the propagation and synthesis of foreign gene products. The gene of interest is co-transfected with linearized baculovirus DNA into the clonal tissue culture line Sf9 derived from Spodoptera frugiperda insect cells. Originally isolated from ovarian cells, they can be cultured either attached or in suspension using Sf-900 II serum-free media (SFM).
Our lab uses serum-free and supplemented SF-900 II media for the maintenance of Sf9 cells in suspension. Serum-free media is used during transfection whereas supplemented media is used for the maintenance of cultures following transfection.
30ml Sf9 cells at 1 x 10^6 cells/ml and Sf-900 II media, warmed to 27°C. Supplemented Sf900 II media (with 50ml heat inactivated FBS, 2% PSG, 2% L-Glut)
Inoculate a 30ml culture of Sf9 cells (1 x 10^6 cells/ml) with 10ul of sf9 baculoviral supernatant. Transfer cells to an incubator, shaking overnight at 27°C. Split Sf9 cells with the Sf-900 II media to maintain them at 2 x 10^6 cells/ml . The cell culture should be maintained at 2 x 10^6 cells/ml until the cells stop dividing. Once this point is reached, the culture should be incubated, shaking for a further 3-5 days. Supernatant can then be harvested for recombinant protein production (in our lab, using a secondary transfection with High-Five insect cells).
The Sf-900 supplemented media should be filtered through a 0.2um filter prior to use and refrigerated when not in use. Media should be warmed gently to 27°C prior to use in cell cultures. Cell culture flasks should not be sealed – if using screw top flasks they must be loosened slightly to prevent sf9 cell death.
Sf-900 II media is a media that can be used serum-free during transfection phase and supplemented during the expansion phase of recombinant protein production using sf9 insect cells.
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Product Name: Sf-900 II Serum-Free MediaSupplier Page