Stage 3 of 3: Generation of Stable, Transfected Cell Lines: Selection

This final video of our three-part series about generation of stable, transfected cell lines demonstrates selection of successfully transfected cells which are resistant to the selection antibiotic. In this video, we use the selection antibiotic G418 Disulfate (Geneticin) in combination with CHO cells. This is the third and final step of generating stable, transfected cell lines.
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[@mortezahosseini2426]

Great video. A quick question. Do you still keep selection antibiotic (here G418) during single cell colony formation and expansion in culture flasks?

[@toku-ecompany7075]

Thank you for question. Yes, we keep the cells in selection medium until we have enough cells for freezing (to ensure high purity of cell line stock). Later on the cells can be cultured without the selection antibiotic, however, they should be monitored for expression of gene of interest. If the percentage of positive cells drops during the selection-free culture, the selection antibiotic is re-introduced.

[@ThanhNguyen-bs3mn]

How do you prepare/collect Conditioned selection medium?

[@toku-ecompany7075]

Conditioned selection medium is prepared by adding the following components to the fresh culture medium:
- The conditioned medium - the medium which was collected from the cultured cells - to achieve the concentration
of 10 % (so e.g. 10 ml of the conditioned medium added to 90 ml of the fresh medium)
- The selection antibiotic to reach the concentration determined in a kill curve experiment - explained in the video
‘Generation of Stable, Transfected Cell Lines: Kill Curve (Stage 1 of 3)’.