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Methods Mol Biol. 2012;889:181-95. doi: 10.1007/978-1-61779-867-2_11.

Mouse embryonic stem cell adherent cell differentiation and cytotoxicity assay.

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National Health and Environmental Effects Research Laboratory, Research Triangle Park, NC, USA.


There are thousands of environmental chemicals for which there is limited toxicological information, motivating the development and application of in vitro systems to profile the biological effects of xenobiotic exposure and predict their potential developmental hazard. An adherent cell differentiation and cytotoxicity (ACDC) assay was developed using pluripotent mouse embryonic stem cells (mESCs) to evaluate chemical-induced effects on both stem cell viability and differentiation. This assay uses an In-Cell Western technique after a 9-day culture. DRAQ5/Sapphire700 cell/DNA stains are used to quantify cell number and myosin heavy chain (MHC) protein is used as a marker of cardiomyocyte differentiation. MHC is corrected for cell number, thereby separating cytotoxicity and effects on differentiation. The ACDC assay can be used to evaluate the effects of xenobiotics on mESC differentiation and cell number in the same sample.

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