Figure 5.

Effect of glucose oxidase on sham-transfected HuH7 cells. (A) MTT assay. 15 × 10⁴ cells per well were seeded in 96-well plates, incubated overnight, then treated with 75 mU ml⁻¹ glucose oxidase for 2 h. MTT uptake by viable cells was assessed by the addition of 50 μg MTT per well for 4 h. Plates were read at 570 nm following further overnight incubation with 100 μl per well of 10% SDS, pH 3.0. Glucose oxidase treatment resulted in a reduction in the viable cell population to 38% of untreated cells. (B) Gating of live and dead cells by forward- and side-scatter on flow cytometry. Cells were seeded in six-well plates (1 × 10⁶ cells per well), incubated overnight, treated for 2 h with 75 mU ml⁻¹ glucose oxidase, then harvested. Following two washes in PBS, flow cytometry was performed on a Coulter Epics XL flow cytometer. Treatment with glucose oxidase resulted in a marked shift in the cell population, increasing mean density and decreasing size. The resultant populations were subsequently gated as live and dead. The spread of cells into the dead gate in untreated samples was constant throughout our experiments, but not observed in untransfected HuH7s, and was considered to represent an effect of ongoing selection with G418. (C) Quantification of live and dead gates revealed that GO treatment resulted in a 65% shift from live to dead gates.