ES Cell Subcloning Protocol
NIH ，Stem Cell Unit
胚胎干细胞亚克隆
Subcloning Protocol
We use an adapted form of the feeder-free protocols detailed in Xu et al. (Nature Biotechnology 19:971–974, 2001) and outlined below.
- Conditioned medium is prepared by incubating mitotically inactivated MEFs with hESC medium at a ratio of 1ml per 100,000 MEFs. This medium is collected every day up to 10 days and may be stored frozen for up to 1 month.
- For subcloning plates, mitotically inactivated MEFs are plated at a density of 0.75 x 105/ml - 1.5 x 105/ml with 2.5ml per well of a gelatin-coated 6-well dish.
- hESC are collected by sedimentation, washed once in PBS, and dissociated with Trypsin-EDTA (Invitrogen Cat #25300-054) or prewarmed Cell Dissociation Buffer (Invitrogen Cat #13150-016). Cells are centrifuged at 200g for 5 minutes, resuspended in conditioned medium containing 8ng/ml bFGF, counted, and serially diluted for plating at a concentration of 100 cells per well.
- Conditioned medium is replaced every day until colonies begin to appear (3 to 7 days), at which time normal hESC medium containing 4ng/ml bFGF is used.
- The number of colonies formed is assessed on day 7 of culture (cloning efficiency).
- When colonies reach approximately 100 to 200 cells, they are individually transferred to new wells.
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