Corneal cups were washed once with PBS and moved (with the culture adaptor) into a Plexiglass incubator warmed to 37°C and gassed with 5% CO2. One hundred microliters of a bicarbonate-rich Ringer's solution (BR) was pipetted into the endothelial concavity. The BR consisted of (mM) 150 Na+, 4 K+, 0.6 Mg2+, 1.4 Ca2+, 118 Cl−, 1 H(PO4)2−, 10 HEPES, 28.5 gluconate, 5 glucose, and 28.5 NaHCO3 −. A 10-μL sample was taken, saved for lactate assay, and replenished with 10 μL fresh BR every 5 minutes for 30 minutes. After washing with PBS once, central cornea thickness (CCT) was measured using an E.T.-1 contact lens thickness gauge with 1-μm resolution (Rehder Development Co., Castro Valley, CA, USA). The cornea was then trephined to a 10-mm central button and immediately placed in a mortar, submerged in liquid nitrogen to stop further metabolism, and pulverized using a pestle. The powder was then collected in an Eppendorf tube filled with 0.5 mL PBS, vortexed vigorously for 30 seconds, and centrifuged at 1800g for 15 minutes. The supernatant was collected for lactate assay, and the pellet was retained for assay standardization. The pellet was dried in a vacuum centrifuge for 2 hours at 30°C and then weighed. Lactate concentration was determined using a lactate assay kit from BioVision Research Products (Milpitas, CA, USA) and represented as nmol lactate/mg dry tissue.