Cell-Tak coating interferes with DNA-based normalization of metabolic flux data.

Metabolic flux investigations of cells and tissue samples are a rapidly advancing tool in diverse research areas. Reliable methods of data normalization are crucial for an adequate interpretation of results and to avoid a misinterpretation of experiments and incorrect conclusions. The most common methods for metabolic flux data normalization are to cell number, DNA and protein. Data normalization may be affected by a variety of factors, such as density, healthy state, adherence efficiency, or proportional seeding of cells. The mussel-derived adhesive Cell-Tak is often used to immobilize poorly adherent cells. Here we demonstrate that this coating strongly affects the fluorescent detection of DNA leading to an incorrect and highly variable normalization of metabolic flux data. Protein assays are much less affected and cell counting can virtually completely remove the effect of the coating. Cell-Tak coating also affects cell shape in a cell line-specific manner and may change cellular metabolism. Based on these observations we recommend cell counting as a gold standard normalization method for Seahorse metabolic flux measurements with protein content as a reasonable alternative.

Metabolic cooperation in the ovarian follicle.

Granulosa cells (GCs) are somatic cells essential for establishing and maintaining bi-directional communication with the oocytes. This connection has a profound importance for the delivery of energy substrates, structural components and ions to the maturing oocyte through gap junctions. Cumulus cells, group of closely associated GCs, surround the oocyte and can diminished the effect of harmful environmental insults. Both GCs and oocytes prefer different energy substrates in their cellular metabolism: GCs are more glycolytic, whereas oocytes rely more on oxidative phosphorylation pathway. The interconnection of these cells is emphasized by the fact that GCs supply oocytes with intermediates produced in glycolysis. The number of GCs surrounding the oocyte and their age affect the energy status of oocytes. This review summarises available studies collaboration of cellular types in the ovarian follicle from the point of view of energy metabolism, signaling and protection of toxic insults. A deeper knowledge of the underlying mechanisms is crucial for better methods to prevent and treat infertility and to improve the technology of in vitro fertilization.