Enzchek phospholipase a2 kit

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.

Phospholipase A₂ (PLA₂) activity was carried out in accordance with the manufacturer’s protocol (EnzChek Phospholipase A2 kit; E10217, Invitrogen, Waltham, MA, USA) and our published protocol [3 (link),81 (link),83 (link)]. Briefly, SRA-hLECs and mLECs were treated with different concentrations of Hyd for 24 h. Thereafter, total protein was isolated and quantified by BCA protein assay (ThermoFisher Scientific, Waltham, MA, USA). To prepare a standard curve, different concentrations (0–10 Units/mL) of PLA₂ was made by diluting PLA₂ stock solution (500 Units/mL) with 1 × reaction buffer up to 50 µL. For sample preparation, an equal amount of protein was diluted with 1 × PLA₂ reaction buffer to make volume up to 50 µL. The reaction was started by adding 50 µL of the substrate-liposome mix to each microplate well consisting of control, standard, and the samples with 100 µL total reaction volume. The fluorescence units were measured at optical density (O.D.), Ex485 nm/Em535 nm, using a microplate reader (DTX 880, Multimode Detector, Molecular device, San Jose, CA, USA).

Phospholipase A2 activity was measured according to the manufacture's protocol (EnzChek Phospholipase A2 kit; E10217, Invitrogen). In brief, LECs transfected with different plasmid constructs were harvested and cell lysates were isolated. Proteins were measured (Bradford method) and normalized with GFP reading. For standard curve, PLA₂ stock solution (500 units/ml) diluted with 1 × reaction buffer to make different concentration (0–10 units/ml) of PLA₂. For sample, equal amount of protein were diluted with 1 × PLA₂ reaction buffer up to 50 μl volume, then 50 μl of the substrate-liposome mix were added to each microplate well containing standards, controls and samples to start the reaction with total volume 100 μl. The fluorescence of each well was measured at Ex485 nm/Em535 nm using microplate reader (DTX 880, Multimode Detector, and Molecular Device) and presented.