What are suitable positive and negative controls for tube formation experiments?
Short answer: In tube formation experiments, a positive control is a condition where cells reliably form tubes (e.g., VEGF-treated endothelial cells on Matrigel®), while a negative control is a condition where little or no tube formation is expected (e.g., inhibitor-treated cells, solvent control, or non-permissive matrix).
Definition of Positive and Negative Controls
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).
Selection of Suitable Controls
The optimal positive and negative control for tube formation experiments within the ibidi Angiogenesis labware strongly depends on the cell type, 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.
Examples of Positive and Negative Controls
In the following, you find possible approaches for positive and negative controls in tube formation assays:
- 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.
- 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.
- 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.
- 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.
- If the cells are treated with any dissolved substance (e.g., in DMSO or ethanol), use the solvent only as a negative control.
Key Considerations for Tube Formation Assays
- The choice of controls depends on the cell type, matrix, and experimental setup.
- Growth factors in standard media can influence baseline tube formation.
- Matrix composition strongly affects angiogenic behavior.
- Literature validation is recommended for reproducible results.
Recommended ibidi Solutions for Tube Formation Assays
For reproducible and standardized angiogenesis assays, ibidi provides both detailed application resources and labware designed for controlled cell culture conditions and reliable imaging:
- Angiogenesis Application Chapter with protocols, background information, and experimental guidance
- µ-Slide 15 Well 3D for defined 3D tube formation assays in hydrogels
- µ-Slide 96 Well 3D for higher-throughput angiogenesis experiments
These resources and systems support consistent matrix handling, defined geometry, and reproducible imaging conditions for tube formation and angiogenesis studies.