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.
Autologous and allogenic human pericardia used as biomaterials for cardiovascular surgery are traditionally crosslinked with glutaraldehyde. In this work, we have evaluated the resistivity to collagenase digestion and the cytotoxicity of human pericardium crosslinked with various concentrations of glutaraldehyde in comparison with pericardium crosslinked by genipin, nordihydroguaiaretic acid, tannic acid, and in comparison with unmodified pericardium. Crosslinking retained the wavy-like morphology of native pericardium visualized by second harmonic generation microscopy. The collagenase digestion products were analyzed using SDS-PAGE, capillary electrophoresis, and a hydroxyproline assay. Glutaraldehyde and genipin crosslinking protected the native pericardium efficiently against digestion with collagenase III. Only low protection was provided by the other crosslinking agents. The cytotoxicity of crosslinked pericardium was evaluated using xCELLigence by monitoring the viability of porcine valve interstitial cells cultured in eluates from crosslinked pericardium. The highest cell index, reflecting both the number and the shape of the monitored cells was observed in eluates from genipin. Crosslinking pericardium grafts with genipin therefore seems to be a promising alternative procedure to the traditional crosslinking with glutaraldehyde, because it provides similarly high protection against degradation with collagenase, without cytotoxic effects.
Diffraction is analyzed for oblique propagation of light beam under a large angle. Fresnel diffraction approximation is valid provided the beam is deflected into the direction of oblique propagation, the structure of the diffraction screen is projected onto the plane perpendicular to the propagation direction, and the diffraction pattern is observed in the plane perpendicular to the propagation. The task is illustrated by the diffraction due to a circular aperture. and Je analyzována difrakce pro šikmé šíření světla pod velkým úhlem. Přiblížení Fresnelovou difrakcí je platné za předpokladu , že svazek je odchýlen do směru šikmého šíření, struktura difrakčního stínítka je promítnuta na rovinu kolmou ke směru šíření a difrakční obrazec je pozorován na rovině kolmé k šíření. Úloha je ilustrována difrakcí na kruhové apertuře.