To quantify ALB and AFP secretion, organoid culture supernatants were collected after 24 hours and the concentrations of secreted ALB and AFP were measured using Human Albumin (Bethyl Laboratories) and Alpha-fetoprotein (Abcam) ELISA Kits, according to the manufacturers’ instructions. Absorbance was measured using a Spectra Max M3 microplate reader (Molecular Devices).
Human albumin
Manufactured by Fortis Life Sciences
Sourced in United States, Belgium
Human albumin is a protein found in human blood plasma. It serves as a crucial component in maintaining the body's fluid balance and transporting various molecules throughout the circulatory system.
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Lab products found in correlation
Alpha fetoprotein, by Abcam (1 mentions)
Spectramax m3, by Molecular Devices (1 mentions)
Mrx microplate reader, by Dynex (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Ammonium chloride, by Merck Group (1 mentions)
Lsr 2 flow, by BD (1 mentions)
Cytofix cytoperm method, by BD (1 mentions)
Human igm, by Jackson ImmunoResearch (1 mentions)
Hil 7, by Miltenyi Biotec (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Fortessa, by BD (1 mentions)
Human lyve1, by Agilent Technologies (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
6 protocols using human albumin
1
Quantifying ALB and AFP Secretion
2
Serum Biomarker Quantification in Mice
3
Evaluating Hepatocyte Response to AAT
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To determine if AAT (2–8 mg/ml) affected hepatocyte viability and function, MTT, albumin and urea assays were carried out. AAT was dissolved in 100 μl William's E (WE) medium and added to the cell culture media. Hepatocyte viability was determined using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Briefly, cells were cultured for 24 h at 37 °C, with 5% CO2, supernatant was removed and cells were cultured with serum-free medium containing 0.5 mg/ml of MTT (Sigma-Aldrich, Dorset, UK) for 4 h. After removal of the MTT, the produced formazan was dissolved in DMSO and the optical density read at 570 nm on a Dynex MRX microplate reader. For albumin quantification, cell culture medium was collected 12 h post-plating and enzyme immunoassays carried out for human albumin (Bethyl Laboratories, Inc., TX, USA). Ammonia metabolism was measured using a QuantiChrom™ Urea Assay kit (Universal Biologicals, Cambridge, UK). Cells were washed with PBS and incubated with 5 mM ammonium chloride (Sigma-Aldrich, Dorset, UK) for 6 h before measurement of urea synthesis.
4
Quantification of Plasma Immune Components and T Cell Functional Assay
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Human albumin (cat# E80–129 Bethyl Laboratories), human IgM (cat# 109–035–043 Jackson ImmunoResearch), and human IgG (cat# 109–035–008 Jackson ImmunoResearch) levels were quantified in the plasma by ELISA according to the manufacturer’s instructions and previously described protocols [16 (link)]. Mononuclear cells from blood, spleen, bone marrow, thymus and lymph nodes were isolated as previously described [16 (link)]. Liver leukocytes were isolated after eliminating circulating cells by perfusing the liver with PBS in situ through the vena cava as previously described [28 (link)]. To test the functionality of T cells, splenocytes were cultured in IMDM with 10% fetal calf serum, 10 IU/ml hIL-2 (Chiron), 100 ng/ml hIL-7 (Miltenyi), 4x105 Dynabeads CD3/CD28 (Life technologies) for 9 days then stimulated for 4 hours with 50 ng/ml PMA and 1 microg/ml ionomycin for 4 hours prior to intracellular cytokine staining using the BD cytofix/ cytoperm method (BD biosciences). Flow cytometry was performed with directly conjugated antibodies according to standard techniques using a Fortessa and LSRII flow cytometers (Becton Dickinson). LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen) and a 405 nm excitation were used to exclude dead cells. The list of anti-human antibodies used in flow cytometry is detailed in S1 Table . Analysis was performed with Flowjo Version 8.8 (TreeStar).
5
Quantifying Mouse Albumin Levels
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human albumin levels in mouse sera were determined using the human albumin enzyme-linked immunosorbent assay (ELISA) kit (Bethyl Laboratories; catalog no. E88-129).
6
Immunofluorescence and Histological Analysis of Liver and Spleen
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Cryosections were made of the liver and spleen by embedding the tissue in Tissue-Tek OCT medium (Bayer). Sections of 5–6 µm were cut, affixed to poly-L-lysine–coated glass slides and kept at −20 °C before use. Sections were stained for human Lyve1 (Dilution 1/100; DakoCytomation, Heverlee, Belgium) as described earlier3 (link) or human albumin (dilution 1/100 Bethyl Laboratories, Montgomery, TX). Sections were embedded in mounting medium containing DAPI for nuclear staining. Images were taken using Leica SP8 confocal microscope.
For histology, tissues were processed and embedded in paraplast as described previously. Tissues were stained for human Lyve1 (1:100; DakoCytomation, Heverlee, Belgium), CD45 (dilution 1/250, clone HI30; eBioscience, Vienna, Austria) processed and counterstained with hematoxylin and eosin, for the Lyve1 staining only, as described.34 (link) Pictures were taken using a Olympus BX51 microscope.
For histology, tissues were processed and embedded in paraplast as described previously. Tissues were stained for human Lyve1 (1:100; DakoCytomation, Heverlee, Belgium), CD45 (dilution 1/250, clone HI30; eBioscience, Vienna, Austria) processed and counterstained with hematoxylin and eosin, for the Lyve1 staining only, as described.34 (link) Pictures were taken using a Olympus BX51 microscope.
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