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Research TopicsStem Cell Research at NIHNIH Stem Cell UnitSubcloning Analysis
Subcloning Analysis
View the subcloning protocol
| Cell Line |
MEF density |
# cells
plated |
# clones
after 7 days (%) |
# clones
cultured |
| BG01(1) |
0.375 x 106/well
6 well plate |
100/well
2 wells |
45 (~23%) |
11 |
|
0.187 x 106/well |
140/well
6 wells |
62 (~7.4%) |
|
| TE03 |
0.187 x 106/well
6 well plate |
200/well |
97 (~8%) |
8 |
| TE06 |
0.375 x 106/well
6 well plate |
200/well
6 wells |
8 (~1%) |
2 |
| UC06 |
0.187 x 106/well
6 well plate |
100/well
6 wells |
67 (~11%)
13 (~2%)
13 (~2%)
56 (~9%)
|
6
4
4
NA
|
| WA01 |
0.187 x 106/well
6 well plate |
100/well
6 wells |
4 (<1%) |
3 |
| WA07 |
0.75 x 106/10cm dish |
125/dish
4 dishes |
23 (~5%) |
12 |
| WA09 |
0.187 x 106/well
6 well plate |
100/well
6 wells |
141 (~23%) |
5 |
NA = Not applicable
For most cell lines, these numbers represent a single experiment. Replicates are in progress.
- BG01(1)—There appear to be two distinct morphologies that vary in their immunoreactivity to SSEA-1 antibody. One clone has tightly packed cells and is SSEA-1–negative, whereas the other has more flattened, larger cells and is SSEA-1–positive. There was no difference in other undifferentiated marker expression.
- WA01—No difference in morphology, immunostaining with undifferentiated markers or Oct4 FACS analysis.
- WA07—No difference in morphology or in immunostaining with undifferentiated markers.
- WA09—No difference in morphology or in immunostaining with undifferentiated markers.
- UC06(1)—(in progress)
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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.