A method to quantify apoptosis in mouse embryos using flow cytometry

Abstract

Cell proliferation, differentiation and apoptosis play a major role in morphogenesis and organogenesis of the developing embryo. Teratogens can cause alteration in apoptotic patterns in the developing embryo which may lead to fetal malformations; however, no studies have quantified apoptosis in early embryos. We developed a protocol to quantify apoptosis in CD‐1 mouse embryos using flow cytometry. Valproic acid (VA) and diabetes mellitus were then used to induce neural tube defects and cardiac malformations respectively to evaluate apoptotic changes induced by known teratogens. Apoptotic levels were quantified in head regions of VA exposed embryos and hearts of hyperglycemic fetuses. Embryonic cells were disassociated enzymatically followed by differential staining with Annexin‐V and propidium iodide to differentiate apoptotic, dead and live cells. Apoptosis levels increased in the head regions of VA exposed embryos (18.1± 2.2 %) than the controls (12.7 ± 1.9 %). Increased levels were also observed in the hearts of hyperglycemic fetuses compared to controls. TUNEL assay, used to localize apoptotic regions, showed increased apoptosis along the open neural folds of the VA exposed embryos. This is the first report using flow cytometry to evaluate apoptosis in early embryos, and the results indicate this may be a useful method to quantify relative changes in apoptotic levels in teratogen exposure.Grant Funding SourceNIH grant K01RR16241‐01e