Finnigan surveyor system

After the 24-h treatment with PLEE, cells were harvested with trypsin (0.05%), washed with phosphate buffered saline (PBS) and stored at − 20 °C until lactose extraction, and 0.5 mL of medium were collected for further analysis. Cells and medium were lysed by 4 rotations of freezer and thaw cycles followed by 30 min of sonication. Then, cells were centrifuged in 350 g for 5 min and 0.5 mL of upper phase was collected and filtered in 0.45 µm (PES) and stored in − 20 °C until injection for HPLC analysis. Lactose was separate on a silica column in a Rezex-ROA-acids H⁺ (8%) 150X78 mm column, as previously described by Tayeh et al.^{63 (link)}. HPLC analysis with binary gradient elution on a Thermo scientific Finnigan Surveyor system equipped with a refractive index detector at 68 °C. Sulfuric acid (0.005 N) was used for elution at flow rate of 0.6 mL/min for 14 min; the injected volume was 20 µL. Standard curves for lactose (Sigma Aldrich, Israel) created after injection in concentrations of 50, 100, 200, 500, 1000, 2000 and 5600 ppm. Identification and quantification were determined by establishing a calibration curve of external standard of known lactose concentration dissolved in water. The calibration curve for lactose had an R² = 0.99.

GSH levels in lens tissues were measured according to an HPLC method developed within our laboratory and described previously [30 (link), 43 (link)]. Briefly, each lens was homogenized in a serine borate buffer (pH 7.8), followed by centrifugation at 5000 × g for 10 min at 4 °C. The resulting supernatant was removed and diluted. A 50 μL aliquot of this diluted supernatant was then added to 200 μL of nanopure water. To each sample, 750 μL of 1 mM NPM in acetonitrile was added. The derivatization reaction was allowed to proceed at room for 5 min, after which time 10 μL of 2 N HCl was added to quench the reaction. Each sample was transferred to an HPLC vial through a 0.45 μm pore filter. HPLC separation and analysis were performed on a Finnigan Surveyor system (Thermo Scientific), which included an Auto Sampler Plus, LC Pump Plus, FL plus Detector, and a 250 × 4.6 mm Reliasil ODS-1 C₁₈ column with 5 μm packing material (Orochem Technologies Inc., Naperville, IL, USA). This system was used for all subsequent HPLC analyses described herein. The mobile phase was composed of acetonitrile and nanopure water (70:30 v/v ACN: H₂O) with 1 mL/L acetic acid and 1 mL/L phosphoric acid added to the water to adjust the pH to 2.5. An isocratic method was used, with a flow rate of 1 mL/min. Excitation and emission wavelengths for NPM derivatives were set at 330 and 376 nm, respectively.