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查看更多产品信息 FreeStyle™ CHO Expression Medium - FAQs (12651022, 12651014)
3 个常见问题解答
在使用FreeStyle 293表达培养基时，我们推荐稍早一些收获细胞，以减少宿主细胞蛋白所造成的非特异性结合，之后对培养上清液进行0.2 或0.1 µM的膜过滤，再上样至镍柱。另一方面，FreeStyle CHO表达培养基中含有的EDTA将会对镍柱起到洗脱的作用。因此在上样之前，应对这一培养基进行透析或缓冲体系交换，也可通过向上清液加入1-3 mg/L NiSO4或NiCl来螯合EDTA。另外可使用铁盐或钴盐。
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.
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 clumps do form, you can try the following protocol to select for cells that don't form clumps:
- Transfer cells into an appropriate size centrifuge tube that will hold the entire cell suspension.
- Allow cells to sit undisturbed for about 5 minutes. The time can vary depending on the specific cell line. Larger cell clumps will settle to the bottom of the tube.
- Collect cells from the upper portion of the tube to passage into a new flask.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.