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Angiogenesis Technical Support

TCS Cellworks has developed and provided cell-based research products for both academic and industrial scientists for many years. During the development and ongoing use of AngioKit™, we have gained extensive experience in the field of in-vitro angiogenesis research. We are happy to share our experiences and to provide in-depth technical support - from helping you to get a new project started, to providing trouble-shooting advice along the way. Please do not hesitate to contact us with your technical questions - we guarantee that you will be able to discuss your project with an experienced scientist. We can be contacted as follows:

Tel: +44 (0) 1280 827475

Fax: +44 (0) 1280 827477

Email: office@tcscellworks.co.uk

Online request form

You may also find useful technical resources in the "About AngioKit™" section of this website, and the Frequently Asked Questions (below).

Contract Research

We offer a range of contract research services using AngioKit™ and related in-vitro products. Please see the Contract Testing section of our main website for further information, and feel free to contact us to discuss your requirements.

Frequently Asked Questions / Trouble Shooting Guide (AngioKit™)

Transport:

Is AngioKit™ adversely affected by transport to our lab?

AngioKit™ is shipped at ambient temperature in a protective box and outer case, and has proven to be remarkably robust to transportation. Under normal conditions, 3 day shipment periods cause nor adverse effects to performance. Although it has been known for a kit to recover from a 7 day shipment period and perform adequately, this is an exceptional case and transit times of 4 days or longer can cause irreversible damage.

The central areas of some wells are devoid of cells. Why is this?

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

Fibroblast morphology appears shortened and there are clearly defined spaces between cells. What is the cause?

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

Why has the normal spindle morphology disappeared?

AngioKit™ can tolerate up to 6 hours at 4-6°C with a suitable recovery period, but damge 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™ in your laboratory:

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

Customers must examine AngioKit™ immediately upon arrival, following subaseal removal, under a light microscope. Any observations concerning damaged products should be reported straight away; if possible supporting photographic images are helpful for trouble shooting.

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

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

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?

This is caused by addition of optimised AngioKit™ 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.

Use of AngioKit™:

How should test articles be prepared for application to AngioKit™?

Preparation of test articles needs to be conducted bearing in mind that AngioKit™ is 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 the 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.

No tubule formation occurred. Why?

There are many possible reason 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.

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

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 Day 11. If the sheet is still partially attached to the well, attempt to gently fix and stain the tubules.

Why are cells floating in the well?

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.

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

It is important to act promptly to contain the contamination. Use your laboratory’s preferred method for this, or contact us for advice.

Staining AngioKit™:

No tubule formation can be observed. Why?

Check that antibodies were added in the correct order.

What causes high background staining?

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.

What causes faint staining of the tubules?

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:

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.