Human albumin elisa kit

The following substances and reagents were used in the experiments without additional purification: 300 Bloom gelatin of Type A derived from porcine skin collagen, urea (ultra-pure grade), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide) (EDC), and ammonium chloride—all from Sigma-Aldrich Inc. (St. Louis, MO, USA); a 40% aqueous solution of glyoxal (GXL) and a 96% aqueous solution (v/v) of ethanol—both from Reakhim (Moscow, Russian Federation). Dulbecco’s modified Eagle’s medium (Nutrient Mixture F-12 (DMEM/F12), alanyl–glutamine and Giemsa dye were purchased from PanEco (Moscow, Russian Federation). Fetal bovine serum (FBS), antibiotic/antimycotic, and HEPES were purchased from Gibco Inc. (Billings, MT, USA). Basic fibroblast growth factor (bFGF) was purchased from Peprotech (Cranbury, NJ, USA). A Human Albumin ELISA Kit, Picogreen QuantiT™, Live/Dead^® Cell Viability/Cytotoxicity Kit and PrestoBlue™ Cell Viability Reagent were purchased from Invitrogen Corp. (Carlsbad, CA, USA). DNeasy Blood & Tissue Kit was obtained from QIAGEN (Hilden, Germany). A neodymium-containing contrasting solution for scanning electron microscopy (SEM) was obtained from Glaucon LLC (Moscow, Russian Federation). All aqueous solutions were prepared using deionized water of Milli-Q quality generated with a Simplicity Water Purification System (Millipore, Molsheim, France).

Cryopreserved PHH from single donors (BD Gentest, Tewksbury, MA; Lonza, Walkersville, MD; and Thermo Fisher Scientific) were used for all in vitro experiments. PHH were thawed according to the supplier’s instructions using Cryopreserved Hepatocyte Recovery Medium (CM7000; Thermo Fisher Scientific), resuspended in Williams’ Medium E (MilliporeSigma, Burlington, MA) with plating supplements (CM3000; Thermo Fisher Scientific), and plated at a density of 50,000 cells/cm². One day after seeding, PHH were serum starved overnight in serum-free Williams’ Medium E with maintenance supplements (CM4000; Thermo Fisher Scientific). Cells were then incubated for 48 h in DMEM supplemented with 0.5% BSA (free fatty acid free; MilliporeSigma) and 1% penicillin–streptomycin with or without the addition of 400 μM sodium oleate (68 (link), 69 (link)).
For albumin measurements, media were collected at the end of 48 h of lipid loading, and albumin concentration was measured with a commercially available human albumin ELISA kit (Invitrogen, Waltham, MA).
After 48-h lipid loading, media were changed to DMEM with 1% penicillin–streptomycin with or without the addition of cytoskeletal drugs: 5 µM blebbistatin (MilliporeSigma), 5 µM latrunculin (MilliporeSigma), or 10 µM nocodazole (Cayman Chemicals, Ann Arbor, MI). Cells were treated for 4 h before fixation.