Table 5.
Troubleshooting Table
| Step No. | Problem | Possible Reason | Solution |
|---|---|---|---|
| 1A(iv), 1A(vi) 1B(iii) | Cultures growing too slowly | Not enough viable cells | Increase the amount of starter inoculum. |
| 1A(iv), 1A(vi) 1B(iii) | Cell death | Contamination | Start fresh from primary cultures and follow sterile procedures. |
| Incubator conditions (aeration, pH, temperature and lighting) not optimal | Loosen the cap/foil to allow aeration, monitor CO2 levels. For Ana cultures, ensure temperature inside incubator does not increase during light cycle due to heat from the lamps and confirm proper exposure of the cultures to light. | ||
| 1A(vii)-(xi), 1B(iv)-(vi) | Poor GV yield | Cultures not confluent | Allow cultures to grow for a few more days. Confluency is determined by the color and turbidity of the cultures as described in the procedure and shown in Figure 2 |
| 1A(xi), 1B(vi) | Incomplete lysis | Dilute the concentrated cell suspension with lysis buffer and/or allow lysis to proceed longer. | |
| 1A(vii)-(xi), 1B(iv)-(vi) | Collapse of GVs | Make sure the cultures are not subjected to agitation above 100 rpm and handle flasks gently while transporting and placing on hard surfaces. | |
| 1A(x), 1B(vi) | Inefficient retrieval of buoyant cells from the separatory funnel | Thoroughly wash the inner walls of the funnel with media/buffer to retrieve the buoyant cells sticking to the sides (especially for Halo). | |
| 1A(xii) | Incomplete separation of GVs from lysate/subnatant | Lysate is too dense, requiring longer spins for GVs to float to the top | Increase time of centrifugation and/or dilute lysate with PBS prior to spin |
| 1A(xii), 1C(v) | Collapse of GVs during spins | Hydrostatic Pressure on GVs greater than critical collapse pressure | Aliquot suspensions into more tubes to reduce height of the column (volume in a single falcon tube should not exceed 40 mL). |
| 1A(xv), 1B(vii) | Pressurization of tubes while opening and closing | Ensure microcentrifuge tubes are closed very gently. The caps of such tubes should not make a snap sound when closed. | |
| 8 | NHS-amine reaction did not work | NHS will hydrolyze in the presence of water/moisture. | Ensure DMSO used for storage is anhydrous. Limit exposure to ambient moisture. Store in a desiccated environment. |
| 4-10 | GVs aggregate after reaction with NHS-moiety | Conjugation of moieties on the GVs destabilizes the protein. | Conjugate fewer moieties on the GV surface by reducing the concentration of NHS-moiety and/or reaction time. |
| Presence of surfactants in the reactant sample. | Purify the NHS-moieties using chromatography or other methods. | ||
| 9, 10 | Subsequent reaction using NHS-moiety conjugated GVs does not work. | Presence of excess NHS-moieties in solution. | Repeat dialysis until excess NHS-moieties are below level of detection. |
| 11-19 | Incomplete GvpC stripping | GV concentration is too high for efficient removal of GvpC | Dilute native Ana GVs in PBS to an OD ~ 5 before adding 10 M urea buffer. |
| 20-38 | Low or no GvpC yield | Low expression of GvpC variant | Check plasmid for mutations, prepare fresh transformations and try reducing temperature and increasing duration of expression. |
| Poor binding to the Ni column | Ensure column is charged and increase incubation time with resin or add more resin. | ||
| Inefficient retrieval of inclusion bodies | Ensure that inclusion pellets are not lost during repeated rounds of centrifugation and resuspension | ||
| 39-41 | Incomplete GvpC re-addition | GV concentration is too high for re-addition reaction to go to completion | Reduce GV OD500 during GvpC re-addition. Confirm re-addition by SDS-PAGE and Coomassie staining. |
| GvpC concentration is too low | Inaccurate quantification of GvpC eluate. | ||
| 39-41 | Protein precipitation is seen after re-addition | Excess unbound GvpC forms precipitates in PBS. | Reduce stoichiometric excess factor of GvpC during re-addition. |
| 42-45 | No fluorescence from SpyCatcher-GFP-labeled GVs | Incomplete SpyTag functionalization | Confirm GvpC-SpyTag re-addition using SDS-PAGE. |
| Incomplete SpyTag-SpyCatcher reaction | Increase excess factor of SpyCatcher during reaction and/or increase reaction time. | ||
| 46A | Collapse curves are inconsistent or noisy | Incomplete collapse | Make sure that there are no air leaks in the connectors or bubbles in the sample |
| Presence of detergents and/or incomplete GvpC addition | Always run control GV samples such as ΔGvpC and GvpCWT to ensure that the re-addition was done under proper conditions and corroborate with SDS-PAGE results. | ||
| 46C | Too much black background on TEM grid | GV sample contains contaminants or phosphate-containing molecules | Using dialysis or centrifugally-assisted purification to resuspend GVs in 10 mM HEPES with 150 mM NaCl buffer. |
| Uranyl acetate precipitate present in solution | Avoid extended exposure to ambient light. Uranyl acetate will precipitate when exposed to light and UV. | ||
| 46C | Too many or too few GVs on the TEM grid | Incorrect GV OD measurement | Measure GV OD and double check the dilution calculation. |
| GVs were not given adequate time to adhere on charged grids. | Excessive wicking can cause too many GVs to come off grids. Reduce how much solution is being wicked off. | ||
| 47-62 | Only the top of the GV sample for in vitro ultrasound phantoms shows contrast | The concentration of GVs is too high. | Lower the concentration of GVs. |
| 47-62 | The signal from GVs and polystyrene beads is very different. | The concentration of GVs or polystyrene beads is too high or too low. | Matching of GVs’ concentration to concentration of beads. |
| 63A, 63B | Weak or no signal after injection | IVC not in the image | Move the transducer to find the optimal location in the IVC and try to align the imaging target with the natural focus of the transducer |
| GVs collapsed prior to or during injection | Check that there is no pressure when injecting through the catheter by manually injecting a small volume of saline with a 30G syringe. If you feel pressure, readjust the catheter into the tail vein and ensure that there are no blocks, allowing the saline to flow smoothly | ||
| Transmit power set too high | Ensure that transmit power does not generate an acoustic pressure on the GVs that exceeds the critical collapse pressure | ||
| 82 | Excessive foam formation | The overall protein concentration in the sample is too high | Reduce the protein concentration – or – add an antifoam agent such as pluronic L81 (ca. 0.1% by vol.); however, such an agent can also have an impact on the integrity of the protein structure |
| 88 | The Xe NMR spectrum does not show any signal or very distorted signals | The excitation pulse is too weak | Increase the amplitude for the excitation pulse |
| The field homogeneity is poor | Perform another shim | ||
| There are still gas bubbles in the sample while acquiring the FID signal | Increase the waiting time after bubbling or consider adding an antifoam agent | ||
| The Rb is oxidized and no hyperpolarized Xe is produced | Check the laser absorption profile of the hyperpolarizer and replace the Rb in the pumping cell if necessary | ||
| 92A(v) | No CEST response from the GVs is observed in the z-spectrum, or the image with on-resonant saturation does not really show a signal loss | GVs have collapsed | Measure the OD of the sample and prepare a fresh one from stock solution if necessary |
| GV concentration too low | Add GVs from stock solution | ||
| Saturation power too low or saturation time too short | Increase the saturation power or the saturation time | ||
| 92A(v) | The saturation response from the GVs is too broad and merges into the direct saturation response | Saturation power is too high | Reduce the saturation power |