Faf bsa

This study uses the fatty acid preparation method of Cousin [19 (link)]. Stock 5mM PA/fatty acid-free bovine serum albumin (FAF-BSA) was prepared as follows. 100 mM PA (Sigma Aldrich, Saint Louis, MO, USA) stock solution was dissolved in 0.1 M NaOH and heated at 70 °C in a water bath. Simultaneously, a 10% (wt/vol) fatty acid-free FAF-BSA (EMD Millipore Corp, Billerica, MA, USA) solution was prepared in deionized H₂O in a 55 °C water bath. To prepare a 5 mM PA/10% FAF-BSA stock solution, 0.5 mL of the 100 mM PA was added to 9.5 mL 10% BSA solution, which was then heated at 55 °C in a shaking water bath for 10 min before it was vortex-mixed for 10 s and then incubated for an additional 10 min in a 55 °C water bath. The PA/FAF-BSA complex solution was cooled to room temperature before being sterile filtered using a 0.45 μm pore size filter. Control for this complex was prepared the same way by using just regular BSA (GE Healthcare Life Sciences, GE Healthcare Life Sciences, South Logan, UT, U.S.A). The prepared 5 mM PA/FAF-BSA and regular BSA aliquoted solutions were stored at −20°C, where they were stable for three weeks. The stored PA/FAF-BSA and its control stock solutions were heated for 15 min at 55 °C and cooled to room temperature before the study.

Human retinal pigment epithelium (RPE) cells (ARPE-19) were purchased from the American Type Cell Culture (ATCC–CRL–2302, Manassas, VA, USA) and cultured in Advanced DMEM/F12 basal medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 20% fetal calf serum (FCS, Merck Life Science S.r.l., Milano, Italy) and 100 U/mL penicillin–streptomycin (Gibco™, Thermo Fisher Scientific, Inc., Waltham, MA, USA), with physiological glucose levels (5 mM). During growth, cells were maintained in a humidified environment (5% CO₂). Further sub-cultures of cells (passage number 12) were performed at the proper density in accordance with each experimental method. To investigate the effect of the high glucose concentrations on ARPE-19 metabolism, cultured cells were treated as previously reported [25 (link)]. A 50 mM concentration of D-Glucose was added to cells for 20 h, followed by the administration of docosahexaenoic acid (DHA) for a further 16 h (Cayman Chemical, Ann Arbor, Michigan, MI, USA—50 mg in 200 µL ethanol), and this was then complexed to fatty acid-free bovine serum albumin (FAF-BSA, Merck Life Science S.r.l., Milano, Italy), prior to use as described [28 (link)], to reach the final concentration of 60 µM of DHA.

A stock solution of PA (Sigma, St. Louis, MO, USA) was prepared by conjugating PA with fatty acid-free bovine serum albumin (FAF-BSA, Sigma), as reported previously [21 ]. In brief, PA was dissolved in Dulbecco's Phosphate-Buffered Saline (DPBS; Welgene Inc., Daegu, Korea) at 60°C for 20 min to make a 20 mM stock solution, and the pH was adjusted to 7.0~7.4 with 1 M NaOH. FAF-BSA was dissolved in DPBS. Next, 20 mM PA solution was diluted in 5% FAF-BSA solution at a ratio of 1:3 (v/v) to generate a 5 mM PA stock solution. Next, PA was diluted in a culture medium to make a 100 μM PA working solution. An 0.08% FAF-BSA solution was used as a control.

Hepatocytes were prepared as described previously^{53 (link)}, with minor modifications. Liver from three each of WT and elovl2 KO salmon per dietary treatment was dissected, quickly perfused via hepatic vein, finely chopped and incubated with 20 ml of Solution A; Hank’s balanced salt solution supplemented with 10 mM Hepes, 1 mM EDTA and 1 mg ml⁻¹ collagenase (Sigma, Missouri, USA) for 45 min at 20 °C. Digested tissues were filtered through 100 μm cell strainer (Sigma) and the cells collected by centrifugation at 400 × g for 3 min. The cell pellet was washed with 20 ml of solution A containing 10 mg ml⁻¹ fatty acid free bovine serum albumin, FAF-BSA (Sigma). Thereafter, the cell pellet was washed with 20 ml freshly prepared solution B (calcium free minimum essential medium supplemented with 100 U/ml Penicillin, 100 μg/ml Streptomycin, 0.25 µg/ml Amphotericin B and pH adjusted to 7.1–7.4 by sodium bicarbonate). The cells were further purified by centrifuging at 400 × g for 30 min on top of 54% percoll solution. The hepatocytes layer were collected and washed twice with solution B.

PA (Sigma, P9767) was conjugated to fatty acid free bovine serum albumin (FAF-BSA; Sigma, A0281). PA (51.2 mg) was dissolved in 10 ml DPBS at 60°C for 20 min to make a 20 mM stock solution, and the pH was adjusted with 1 M NaOH. FAF-BSA was dissolved in DPBS and sterilized by filtering through a sterile membrane filter. PA (20 mM) was complexed in a 1 to 3 molar ratio with 5% FAF-BSA to generate a 5 mM PA stock solution, and then PA was diluted in RPMI-1640 medium to make a 0.4 mM PA working solution. 0.3% FAF-BSA media was used as a control.