Lalistat2

Frozen liver and spleen samples were homogenized in LAL tissue extraction buffer (0.1 M sodium phosphate [pH 6.8], 1 mM EDTA, 0.02% sodium azide, 10 mM DTT, 0.5% NP-40). Protein concentrations were determined with the bicinchoninic acid assay (Pierce, Rockford, IL, USA), using BSA as the standard. LAL activity was determined using 4-methylumbelliferyl palmitate (4-MUP; Gold Biotechnology, St. Louis, MO, USA) as the substrate, as previously described.⁵⁵ (link)^,56 (link) Briefly, 1 μg of protein or 1 μL serum was added to 0.345 mM substrate solution (0.345 mM 4-MUP, 90.9 mM sodium acetate [pH 4.0], 1% [v/v] Triton X-100 and 0.0325% [w/v] cardiolipin), and enzymatic reactions were performed in triplicate in the presence or absence of the LAL inhibitor Lalistat2 (Sigma Aldrich, St. Louis, MO, USA). Reactions were incubated at 37°C for 3 h in the dark. Reactions were terminated by adding 200 μL of 150 mM EDTA (pH 11.5). A standard curve was prepared ranging from 0–33.3 μM 4-methylumbelliferone (4-MU; Gold Biotechnology, St. Louis, MO, USA). Fluorescence was measured on a SpectraMax M2 plate reader (Molecular Devices, San Jose, CA, USA) using a 355-nm excitation filter and a 460-nm emission filter. LAL activity (pmol/min/μg) was calculated by subtracting the enzymatic activity of the inhibited reaction from that of the uninhibited reaction.

Cells were pre-incubated with chemical inhibitors (30 μM of Lalistat 2, 0.2 μM of GSK264220A, 200 μM of Genistein, 10 μg/ml of chlorpromazine, 5 μM of U18666A) for 1 hour and then continuously treated with lipid emulsions for 6 hours.
For the heparin assay, cells were treated with 50 UI/ml of heparin for 2 hours, followed by 2 times washing with PBS. Emulsions were subsequently added to the cells together with the same concentration of heparin.
Human LPL F:1 (Santa Cruz Biotechnology, California, USA) and 5D2 antibodies were added to the cell culture medium 2 hours prior to emulsion loading at the concentration of 2 μg/ml (1:100 dilution).
Genistein, Lalistat 2, chlorpromazine, and heparin were obtained from Sigma-Aldrich, Missouri, USA. GSK264220A was from Tocris (bio-techne), Abingdon, UK. LPL 5D2 antibody was contributed by Dr. Anne Beigneux, Department of Medicine, David Geffen School of Medicine, UCLA, USA.