Rat albumin

Hepatocytes were cultured with the small, medium, or large microbeads, or without microbeads for control experiments until day 21. The albumin secretions produced by the cells were measured. Culture media from the three dishes were collected separately into microtubes 48 h after replacement of the medium. The media were then centrifuged at 8,000 × g for 10 min and the supernatant was transferred to new microtubes. Quantification of the secreted Rat albumin was carried out using an enzyme-linked immunosorbent assay (ELISA) with the two-antibody-sandwich method. Sheep anti-Rat albumin antibody (1.0 µg/well; Bethyl Laboratories, Montgomery, TX, USA) was coated onto each well of a 96-well plate (Nunc, Roskilde, Denmark). After incubating with blocking solution (0.05 M sodium carbonate, pH 9.6), samples were loaded to the wells and incubated for 1 h. Rat albumin (Bethyl Laboratories) was used as a standard. Horseradish peroxidase-conjugated Sheep anti-Rat albumin antibody (2.5 ng/well; Bethyl Laboratories) was then added to each well. Tetramethylbenzidine (TMB) peroxidase substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) was used for the enzyme-substrate reaction, which was stopped by applying 2 M H2SO4. A microplate reader was used to measure the absorbance of the samples at a wavelength of 450 nm.