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LMU | Nikko Brix

Method for determining clonogenic growth in vitro extended


Horst Zitzelsberger and Daniel Samaga from the Research Unit Radiation Cytogenetics, together with researchers from the LMU Hospital and the former Clinical Cooperation Group "Personalized Radiotherapy in Head and Neck Tumors", succeeded in renewing the analysis method of the colony formation test, which is considered the gold standard, and expanding it to include the phenomenon of cellular cooperation. In this way, significantly more valuable measurement data can be obtained.

Analysis of the self-renewing capacity of mammalian cells is done by clonogenic test systems in a variety of disciplines such as medical oncology, stem cell research, cell biology, pharmacology, toxicology and radiation biology. Here, the colony formation assay is the standard method for quantifying the ability of individual cells to grow potentially indefinitely (clonogenically) in vitro.

Protocols for adherent cells (2D analysis) or non-adherent growth (3D analysis in matrices) are available. At this, frequently the phenomenon of cellular cooperation becomes apparent: cells benefit from small volumes and high cell densities, because cellular growth mechanisms gain impact on clonogenic survival. In investigations of growth inhibiting agents on cell models cellular cooperation affects the colony formation and thus the present gold standard (colony formation assay, Franken et al. Nat Protoc 2006).

“We have now further extended this clonogenic growth protocol to include cellular cooperation. The new protocol enables now researchers of various disciplines to analyze clonogenicity regardless of the presence or absence of cellular cooperation”, says Zitzelsberger.

“Mathematically the cooperativity effect can be modelled by linear regression. Applied to experimental data, we use this model to determine the clonogenic survival in dependence from the cellular cooperation. For calculation of the parameters and their statistical uncertainties we provide Excel- and R-Tools”, adds Daniel Samaga, Co-first author of the publication.

His and Zitzelsberger’s findings, developed together with LMU colleagues, were recently published in Nature Protocols.