TABLE 3 |.
Troubleshooting table.
| Step | Problem | Possible reason | Possible solution |
|---|---|---|---|
| 1 | No growth or slow growth of epithelial cells in coculture | Use of a nonrecommended feeder cell line | Use only the J2 strain of mouse Swiss-3T3 fibroblasts |
| 13A(iii) | No live cells after tissue processing | Extended ethanol rinse time | Rinse no longer than 3 s |
| 13B(vi) | No growth of colon or pancreas cell lines | Incubation in an incubator with an atmospheric gas mixture | Incubate in low oxygen (2% O2) |
| 16 | Observed J2 cell proliferation after irradiation | Irradiation dose is too low | Increase irradiation dose (up to 60 Gy) |
| 17 | Low cell viability after freeze-thaw procedure | Storage of cells at −80 °C | Store cells in a liquid nitrogen freezer |
| 20 | Epithelial cells detach together with J2 feeder cells during first step of differential trypsin treatment |
Trypsin treatment was too long | Do not extend trypsin treatment beyond 1 min |
| 25 | Low cell viability after freeze-thaw procedure | Storage of cells at −80 °C | Store cells in a liquid nitrogen freezer |
| 26A(ii) | No colonies visible when recording starts | Cell confluence is too low | Increase the number of cells added to the wells |
| 26A(iv) | No cells visible | Condensation on the plate surfaces | Let the plate adapt to incubator conditions for at least 30 min |
| 26A(vii) | No motility; wrong cell morphology | Temperature, C02 concentration and/or humidity not correct | Make sure that temperature, C02 level and humidity level are within the range for cell growth |
| Box 1 | Low quality of conditioned medium-does not support cell growth | Suboptimal concentration of growth factors | Use the correct number of cells. Condition the medium for no less than 72 h |