Table 2.
Troubleshooting table
| Step | Problem | Possible reason | Solution |
|---|---|---|---|
| 8 and 13; Box 1, step 4 and Box 2, step 7 |
Signal does not increase with respect to time or cell number. Signal reaches a plateau |
A high number of cells are plated or a large volume of conditioned medium is assayed |
When performing the linearity of the reporter assay with respect to time and cell number, the signal might reach a plateau at the highest concentration of cells or at the later time points (especially for SEAP). In this case, the protocol needs to be scaled down either by assaying a smaller volume of the conditioned medium and/or plating lower amount of cells. However, it is not recommended to assay less then 10 μl of conditioned medium as pipetting errors may be higher especially with the Gluc assay |
| 24 and 33 |
Dose-response with respect to drug concentration is not obtained |
Too little or too much drug is added per well |
Higher or lower doses of drug may be needed. In the case of no response, use higher concentration of each drug/well. In the case of high response even with the lowest drug concentration, use less drug/well. |
| 24; Box 3, step 6; Box 4, step 8 |
No reporter activity is observed in blood/serum |
Not enough cells are implanted or transduction efficiency is low |
More cells-expressing the reporter should be implanted. In the case of tumor and other cells that are known to proliferate in vivo, one may choose to wait 1 – 2 weeks to obtain higher reporter signal before starting the drug treatment. |
| 33 | No Gluc activity is observed during bioluminescence imaging |
Not enough cells are concentrated in one location of the animal |
Implant more cells. In the case of using cells which proliferate in vivo, wait 2 weeks before the next imaging session. |
| 33 | Signal obtained from tumors is saturated |
Large tumor size | Decrease the imaging time (10 s – 1min) in order to stay within the linear range of bioluminescence imaging. |
| 33, Box 4, step 8 |
In the case of cells which are known to proliferate in vivo (e.g., tumors) SEAP activity does not increase over time |
The number of cells in vivo is above the linear range of the reporter assay (especially for SEAP). System reached saturation |
Dilute the serum sample in the assay buffer (20-fold dilution rather then 4-fold) by mixing 2 μl of serum with 38 μl of assay buffer and proceed with the SEAP assay |