What would be a suitable positive and negative control for tube formation experiments?

Positive and negative controls are recommended to reduce variables in a tube formation experiment . A positive control is a sample in which the cells are expected to form tubes (depending on the experimental setup), thereby showing the researcher that the assay was conducted properly. A negative control is a sample treated the same way as all other samples but is not expected to show any experimental result (e.g., little or no tube formation).

The optimal positive and negative control for tube formation experiments within the ibidi Angiogenesis labware strongly depends on the used cells, gel matrix, and the general experimental setup. Therefore, we recommend reviewing the literature for successfully used controls, positive and negative, in your topic of interest.

In the following, you find possible approaches for positive and negative controls in tube formation assays:

1. If a specific compound needs to be analyzed for its potency to induce angiogenesis, a sample treated with a known angiogenesis inducer, such as VEGF or FGF2, can be used as a positive control. The concentration strongly depends on the cell type and experimental setup.
2. If primary cells are used, pre-screened endothelial cell lines (e.g., HUVEC), which exhibit a defined reaction upon treatment with specific growth factors, can serve as a positive control. 
3. For some cell types, the ability to form tubes also depends on the gel matrix being used. As a positive control, endothelial cells should show tube formation on growth factor reduced Matrigel^® with starvation medium (a medium without growth factors or serum). Using starvation medium is especially important if pro-angiogenic substances need to be tested because most cell culture media have added growth factors. In order to analyze the real effect of a substance, both the matrix and the medium must be free of any growth factors. As a negative control, the cells can be seeded on a different matrix (e.g., Collagen I), where no tube formation is expected.
4. A tube formation inhibitor (e.g., suramin or sulforaphane) that does not affect cell viability can be used as a negative control. Using this type of inhibitor is especially important if the aim of the experiment is to test an anti-angiogenic substance. 
5. If the cells are treated with any dissolved substance (e.g., in DMSO or ethanol), use the solvent only as a negative control.

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