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Frequently Asked Questions

Please use the information below to troubleshoot any common problems you may have. If you cannot find your question here or the answer is not sufficient then please contact us immediately.

Transportation

Q. Is AngioKit™ and V2a Kit™ adversely affected by transport to our lab?

A. AngioKit™ is shipped at ambient temperature in a protective box and outer case, and has proven to be remarkably robust to transportation. AngioKit™'s are shipped to the UK and Europe with next day express deliveries. AngioKit™'s in transit for 48 hours can display some signs of damage, but recover in most cases.

V2a Kit™ is not a live kit and is much more robust during transportation in comparison to AngioKit™.

Q. In AngioKit™ the central areas of some wells are devoid of cells. Why is this?

A. This phenomenon is caused by delays in transit. Unfortunately, the affected wells rarely recover.

Q. The fibroblast morphology in AngioKit™ appears shortened and there are clearly defined spaces between cells. What is the cause?

A. This is caused by extended cold shock during transport. AngioKit™ may recover after overnight incubation at 37°C.

Q. Why has the normal spindle morphology in AngioKit™ disappeared?

A. AngioKit™ can tolerate up to 6 hours at 4-6°C with a suitable recovery period, but damage becomes too severe if cold shock is longer than this. During winter, shipments in northern latitudes have occasionally been subjected to prolonged cold shock during delivery.

Receiving AngioKit™ or V2a Kit™:

Q. What should I do when the kits arrive at our laboratory?

A. Customers must examine AngioKit™ IMMEDIATELY upon arrival following the AngioKit™ Protocol. Any observations concerning damaged products should be reported straight away; if possible supporting photographic images are helpful for trouble shooting.

A. V2a Kit™ must be unpacked immediately upon arrival. It is important that the cells are transferred directly from the dry ice to liquid nitrogen. Ensure that all components are stored at the correct temperature according to the V2a Kit™ Protocol.

Q. One or more edge wells, and particularly corner wells, of AngioKit™ have a partially stripped cell layer. Why is this?

A. This is caused by over-enthusiastic removal of the subaseal pads used to seal each well during transport. Unfortunately, wells rarely recover.

Q. Following the first medium change, cells detach with areas becoming devoid of cells. Damage may be particularly evident at the side of wells, and some floating dead cells may be seen. What causes this?

A. This is caused by addition of optimised medium too quickly. The problem may occur through the use of a single channel automatic pipette, rather than a serological pipette as recommended. Wells may recover, but this depends on the extent of the damage and wells will not react in the same way across the complete plate.

Q. What is the typical expiry date for AngioKit™ and V2a Kit™'s?

A. AngioKit™'s are live kits and must be processed on receipt.

A.V2a Kit™ has a typical shelf life of 3-6 months. Seeding and growth supplements are supplied frozen to be added to the medium when required.

Use of AngioKit™ and V2a Kit™:

Q. How should test articles be prepared for application to AngioKit™ or V2a Kit™?

A. Preparation of test articles needs to be conducted bearing in mind that AngioKit™ and V2a Kit™ are extremely sensitive to pro- and anti-angiogenic factors. Replacement of the optimized growth medium with significant amounts of buffer or other culture medium and addition of solvents can themselves have a direct effect on the formation of tubules in AngioKit™ as they would in vivo. Current recommendations for solvent use are as follows: Ethanol, dimethylformamide and DMSO can be tolerated up to 0.1%. There is a requirement for at least 80% optimized growth medium (see the kit protocols for more information).

Q. No tubule formation occurred. Why?

A. There are many possible reasons for this, including the following:

1. High concentrations of angiogenic inhibitors have prevented tubule formation. Check control wells.
2. The solvent used to dissolve test compounds may be at a toxic concentration. Run one or more solvent control wells.
3. Cultures have died. This may be due to a number of reasons, including:
   1. Cells were allowed to become dry between medium changes. Aspirate old medium from only a few wells at a time and replace with fresh optimized medium before aspirating the next wells.
   2. Final concentration of solvent, in which treatment is dissolved, is too high. Make a more concentrated stock solution of the test compound and dilute this further in medium.
   3. Ensure all incubations are at 37°C and in a 5% CO2 humidified atmosphere.

Q. What has caused the cell sheet to detach from the well surface?

A. Cultures have been left to grow for too long. Try to fix and stain cultures before this occurs. Contact TCS Cellworks if this problem is encountered before the end of the protocol. If the sheet is still partially attached to the well, attempt to gently fix and stain the tubules.

Q. Why are cells floating in the well?

A. There are two possible reasons for this:

1. This can be the result of adding fresh medium too quickly. Always add fresh medium very gently to prevent dislodging cells. Never use micropipettes for adding medium.
2. Cultures have died. See above for possible reasons.

Q. A small number of wells are contaminated. What can be done?

A. It is important to act promptly to contain the contamination. Use your laboratory's preferred method for this, or refer to Appendix I in AngioKit™ and V2a Kit™ protocols.

Staining AngioKit™ or V2a Kit™:

Q. No tubule staining can be observed. Why?

A. Check that antibodies were added in the correct order.

Q. What causes high background staining?

A. High background staining may occur following overlong exposure to the final staining solution (substrate). To address this problem, colour development may be monitored under a light microscope. The development of a permanent stain only takes a few minutes.

Q. What causes faint staining of the tubules?

A. There are many possible reasons for this, including the following:

1. Antibodies were not added at the correct concentration.
2. Antibody incubation was too short or was performed at the incorrect temperature.
3. Cultures were left to grow for too long. The matrix is preventing adequate penetration of the primary antibody. Leave the primary antibody to incubate at 6°C overnight and the secondary antibody for 8 hours at 6°C before addition of substrate as usual.
4. Staining solutions may have been made up too early. These solutions are light sensitive and must be made up immediately prior to use.

Image Analysis - AngioSys Software:

Q. How does AngioSys Image Analysis software work?

A. The AngioSys software can determine the number of branch points, the number of tubules, the total tubule length and the mean tubule length, or other parameters the investigator wishes to examine.

Q. What computer programmes is the software compatible with?

A. The software is designed for use on a PC running Microsoft Windows 98, ME, 2000 or XP only.

Q. Is there a demonstration version available?

A. A 30 day free trial demonstration version is available. Please contact us for more information or to request your copy. Please note that only one demonstration version is allowed per department or company. After the demonstration period has expired, a full license version must be purchased if you wish to continue using the programme.

Q. I am not happy with the condition of the kit on arrival - how do I contact TCS Cellworks.

A. All contact information can be found from our Contact Us page.

Support from TCS Cellworks is limited to assistance with the AngioSys Image Analysis software, although technical assistance may be available depending on the nature of the enquiry.

Complaints:

Any complaints about the performance of AngioKit™ are dealt with swiftly, and fully investigated. Please contact us.