Eretic2

For each group and time-point a total of four to seven samples were used, depending on availability and quality of sampling, and processed as previously described [68 (link)]. Briefly, weighed (2–8 mg) frozen retina and vitreous/lens tissues were mixed with 70% aqueous methanol and metabolites were extracted by homogenization in a Bullet Blender 24 (Next Advance Inc, Troy, NY, USA) and centrifugation at 16,000× g at 4 °C for 15 min. Extracts were dried under gentle nitrogen stream and reconstituted in 250 μL phosphate buffer (pH 7.4) in deuterated water (D2O) for NMR measurements using a 14.1 T NMR spectrometer. All samples included 0.02 mM of trimethylsilyl-2,3-d4-propionate sodium salt (TSP-d4) as internal standard for quantification.
The collected spectra were processed (phase correction, baseline correction, and referenced to TSP-d4) and the actual concentration of the internal standard in each sample was calculated using a calibrated electronically synthetized digital signal (ERETIC2; Bruker, Billerica, MA, USA). Quantification of absolute concentrations of metabolites was carried out in Chenomx and concentrations were corrected for tissue weight. Three spectra (2 from retina and 1 from vitreous/lens) were excluded from analysis due to insufficient quality.

Supernatants were collected, lyophilized and up-concentrated four times in deuterium oxide (Sigma-Aldrich). 1D proton spectra were recorded at 25°C on a Bruker Ascend 400 MHz Avance III HD equipped with a 5 mm Z-gradient SmartProbe (Bruker). The anomeric proton of α-glucose (5.2 ppm), methyl Hβ of lactate (1.3 ppm) and methylene Hγ of glutamine (2.4 ppm) were integrated and quantified by electronic reference to access in vivo concentrations (ERETIC2, Topspin 3.5, Bruker). The methylamine H of a creatine (3.0 ppm) external standard (Sigma-Aldrich) was defined as the ERETIC reference. Consumption/production was normalized to average number of live cells (average of live cell density when treatment was initiated and live cell density at time of harvest) within the 24h time interval examined to obtain consumption/production /cell/24h. Four independent cultures of Um-Uc-3 and T-24 cells were analyzed for each condition.