PLA2 Activity Assay in Lens and LECs

Phospholipase A₂ (PLA₂) activity was determined according to the manufacturer’s protocol (EnzChek Phospholipase A2 kit; E10217, Invitrogen, Waltham, MA, USA) and our published protocol [75 (link),78 (link)]. In brief, total protein isolated from mouse/human lenses/LECs of different ages or LECs untreated or treated with Metformin as indicated in figures and legends was quantified by Bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Waltham, MA, USA). To prepare the standard curve, different concentrations (0–10 Units/mL) of PLA₂ were prepared by diluting PLA₂ stock solution (500 Units/mL) with 1× reaction buffer up to 50 µL. An equal amount of protein isolated from mouse/human lenses/LECs and LECs was diluted with 1× PLA₂ reaction buffer to make volume up to 50 µL, then 50 µL of the substrate-liposome mix were added to each microplate well-containing control, standard and the samples to start the reaction with 100 µL total volume. The fluorescence units were measured at optical density (O.D.), Ex485nm/Em535 nm using a microplate reader (DTX 880, Multimode Detector, Molecular device, San Jose, CA, USA) as shown in the figures.