Limiting dilution assays were performed to analyze the frequency of cells giving rise to free-floating spheroids (on polyHema) or 3D foci (on tissue culture plastic or on substrates used for studying primary cells)
| Coating | Cell | Plating density (number of cells/well) | |||||||
|---|---|---|---|---|---|---|---|---|---|
| 10 | 20 | 50 | 100 | 200 | 500 | 1000 | 2000 | ||
| PolyHEMA | O53E | 40% | 90% | 100% | 100% | 100% | 100% | 100% | 100% |
| O53P | 50% | 60% | 90% | 100% | 100% | 100% | 100% | 100% | |
| G53E | 90% | 100% | 100% | 100% | 100% | 100% | 100% | 100% | |
| G53P | 50% | 70% | 100% | 100% | 100% | 100% | 100% | 100% | |
| G53AE | 90% | 100% | 100% | 100% | 100% | 100% | 100% | 100% | |
| GEA53 | 100% | 100% | 100% | 100% | 100% | 100% | 100% | 100% | |
| A53E | 0 | 0 | 0 | 30% | 40% | 70% | 100% | 100% | |
| A53P | 0 | 0 | 0 | 10% | 10% | 30% | 70% | 100% | |
| Tissue culture plastic | O53E | 80% | 100% | 100% | 100% | 100% | 100% | 100% | 100% |
| O53P | 50% | 90% | 100% | 100% | 100% | 100% | 100% | 100% | |
| G53E | 100% | 100% | 100% | 100% | 100% | 100% | 100% | 100% | |
| G53P | 50% | 100% | 100% | 100% | 100% | 100% | 100% | 100% | |
| G53AE | 90% | 100% | 100% | 100% | 100% | 100% | 100% | 100% | |
| GEA53 | 80% | 100% | 100% | 100% | 100% | 100% | 100% | 100% | |
| Substrate | O53E | 17% | 67% | 83% | 100% | 100% | 100% | 100% | 100% |
| G53E | 83% | 100% | 100% | 100% | 100% | 100% | 100% | 100% | |
| GEA53 | 50% | 67% | 100% | 100% | 100% | 100% | 100% | 100% | |
Cell dilutions ranged from 10 cells/well to 2000 cells/well in 100 μl aliquots. After 7 d, the fraction of wells containing free-floating spheroids or 3D foci for each cell plating density was calculated.