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

Title:

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

Creator:

Sima, Michal, Martinkova, Stanislava, Kafkova, Anezka, Pala, Jan, and Trnka, Jan

Identifier:

https://cdk.lib.cas.cz/client/handle/uuid:3f0a2885-16ef-4344-ba71-6f43f17ecbe3
uuid:3f0a2885-16ef-4344-ba71-6f43f17ecbe3
doi:10.33549/physiolres.934855

Subject:

metabolic flux analysis, normalization, and Cell-Tak

Type:

model:article and TEXT

Format:

počítač and online zdroj

Description:

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.

Language:

English

Rights:

http://creativecommons.org/licenses/by-nc-sa/4.0/
policy:public

Coverage:

517-526

Source:

Physiological research | 2022 Volume:71 | Number:4

Harvested from:

CDK

Metadata only:

false