To directly test whether Alx3-deficiency is accompanied by increased oxidative stress, we prepared primary mouse embryonic mesenchyme (MEM) cells directly from wild type or Alx3 knockout embryos. These cells normally express Alx3 abundantly30, 32 and are located in the craniofacial region where malformations in Alx3-deficient embryos were found. Using these primary MEM cells we measured ROS production after exposure to low or high concentrations of glucose using fluorescent probes. When cultured in the presence of 5.6 mM glucose, a concentration corresponding approximately to euglycaemic levels in vivo, ROS production levels in cells from Alx3-deficient embryos were not significantly different from those found in control cells from wild type embryos (Fig. 3G). Increasing the concentration of glucose to 30 mM resulted in a significant elevation in the intracellular levels of ROS only in Alx3-deficient cells (Fig. 3G).