Figure 3.

Mitochondrial activity increases with decreasing medium depth. We traced the incorporation of ¹³C₆-glucose into mitochondrial intermediates in hfRPE, measuring the rate of appearance of m+2 labeled intermediates in pooled apical + basal media from 4 to 22 hours after media change (A, C, E), as well as the fraction of each intermediate in the apical versus basolateral media that picked up a ¹³C label at 22 hours (B, D, F). The apical media secretion rate of citrate (A), malate (C), and (E) glutamate derived from glucose (m+2) decreases with higher media volumes. In addition, the fraction of total (B) citrate, (D) malate, and (F) glutamate with a ¹³C label decreases at higher media volumes. These results confirm that limited O₂ diffusion to cells prevents ongoing mitochondrial activity (n = 5–6, donors 3, 4, and 5).