Fig. 1.

Assembly and functionality of the insert-based two-enzyme hypoxia system. (A–F) Commercially available six-well inserts from which the bottom membrane was removed are used as a framework for the assembly of a semipermeable dialysis membrane. (A–D) The basic steps of insert assembly. 1 and 2 in A describe the order in which the steps are performed: 1, insert the semipermeable membrane; 2, insert the plastic ring. (E,F) The assembled insert in a six-well plate. (G) Addition of glucose oxidase and catalase into the upper compartment containing standard cell culture medium leads to the generation of hypoxia in the lower compartment. (H) Schematic depiction of the glucose-oxidase- and catalase-dependent reactions that lead to hypoxic culture conditions. CAT, catalase; GO, glucose oxidase.