Are there any recommendations for preventing or dispersing cell clumps in a suspension culture?

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Answer

When cells meant to be grown in suspension are grown in static culture, they may form clumps. These clumps will severely limit transfection efficiency and protein expression. It is suggested that FreeStyle™ 293 cells in FreeStyle™ media and CHO-S cells in CD-CHO or CHO-SFM are grown in agitated suspension to reduce the appearance of clumps. However, if they do form you can try the following protocol to break up the clumps:

1) Collect all cells in a 50 ml conical tube. Spin at 500 X g for 10 minutes. Remove all media.
2) Resuspend the cells in 5 mls Trypsin-EDTA (0.5% Trypsin, 5.3mM EDTA*4Na). Titurate cells to break up clumps.
3) Spin cells at 500 X g for 10 minutes. Remove all trypsin and resuspend cells in 5 mls CD-CHO media. Titurate cells to break up clumps. Add an additional 20 mls CH-CHO media.
4) Spin cells at 500 X g for 10 minutes. Remove all media and resuspend cells in 5 mls CD-CHO media. Titurate cells to break up clumps. Add an additional 20 mls CH-CHO media.
5) Spin cells at 500 X g for 10 minutes. Remove all media and resuspend cells in 5 mls CD-CHO media. Titurate cells to break up clumps.
6) Count cells and return to culture. Because there is little protein in CD-CHO media it is necessary to remove all trypsin from the cells before returning the cells to culture.

Answer Id: E4097

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f7eb48b3fac4c765e510b2a5cbdaeda5_FAQ

Can FreeStyle™ 293-F cells grown in FreeStyle™ 293 expression medium be transfected with ExpiFectamine™293 Reagent along with enhancers? Is so, will the transfection efficiency be higher than that with FreeStyle™ Max Reagent?

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Answer

You should be able to use the ExpiFectamine™293 Reagent for transfection of Freestyle™ 293-F cells grown in FreeStyle™ 293 Expression Medium; however, the enhancers will provide only a little boost in expression as they are designed to work with higher density cultures that FreeStyle™ 293 Expression Medium cannot support.

Answer Id: E9246

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aed434cccab4d6090f5b8fdaf739833d_FAQ

Can I load the FreeStyle™ 293 Expression Medium or FreeStyle™ CHO Expression Medium directly onto a nickel column for His-tagged protein purification?

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Answer

With FreeStyle™ 293 Expression Medium, we would recommend harvesting the cells a little earlier to reduce any nonspecific binding by host cell proteins and then filtering the culture supernatant through a 0.2 or 0.1 μM membrane before loading onto the column. On the other hand, FreeStyle™ CHO Expression Medium contains EDTA that will strip the nickel column. The medium can be subjected to dialysis or buffer exchange prior to loading on the column, or the EDTA can be chelated by adding 1-3 mg/L NiSO4 or NiCl to the supernatant. Iron or cobalt salts can also be used.

Answer Id: E9250

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