The PDH were isolated from the livers of 1-day-old cherry valley ducklings as previously described [21 (link)]. Briefly, DHBV-negative ducklings were anesthetized with 0.75% pelltobarbitalum natricum (10 mg/kg) and the portal vein was exposed. The liver was perfused with 80 ml of hepatocyte perfusion medium (Invitrogen, Carlsbad, CA, USA) and digested with collagenase IV (Sigma, St. Louis, MO, USA) via the portal vein. After removal of the blade, the liver was dispersed in hepatocyte wash medium (Invitrogen, Carlsbad, CA, USA), and the hepatocytes were separated from the non-parenchymal cells by centrifugation at 50×g. After washing, the hepatocytes were suspended in Leibovitz’s L-15 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 5% FBS (Invitrogen, Carlsbad, CA, USA), 100 units/ml Penicillin-Streptomycin (Invitrogen, Carlsbad, CA, USA), 1 mg/L insulin (Sigma, St. Louis, MO, USA), and 10−5 M hydrocortisone-hemisuccinate (Sigma, St. Louis, MO, USA), and seeded at a density of 1×107cells per 10 cm dish. The PDH were maintained at 37°C without CO2. After 24 h post-inoculation, the PDH were maintained in L-15 medium supplemented with 5% FBS or 1.5% DMSO for 7 d.
Hepatocyte wash medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Hepatocyte Wash Medium is a cell culture medium designed to prepare and maintain primary hepatocytes. It is formulated to wash and suspend hepatocytes during isolation and purification procedures.
Automatically generated - may contain errors
Lab products found in correlation
Liver perfusion medium, by Thermo Fisher Scientific (10 mentions)
Liver digest medium, by Thermo Fisher Scientific (6 mentions)
100 μm cell strainer, by BD (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Liver digestion medium, by Thermo Fisher Scientific (2 mentions)
Liver digest medium, by BD (2 mentions)
Percoll gradient, by GE Healthcare (2 mentions)
Willams e medium, by Thermo Fisher Scientific (2 mentions)
Hydrocortisone hemisuccinate, by Merck Group (1 mentions)
Leibovitz s l 15 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Collagenase b, by Roche (1 mentions)
Facsaria, by BD (1 mentions)
Hepatozyme sfm medium, by Thermo Fisher Scientific (1 mentions)
70 m cell strainer, by BD (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
18 protocols using hepatocyte wash medium
1
Isolation and Maintenance of Duck Primary Hepatocytes
2
Isolation of Mouse Primary Hepatocytes
3
Isolation of Primary Murine Hepatocytes
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Primary hepatocytes were isolated from male mice at 8–12 weeks of age as previously described [21] (link). Briefly, collagenase perfusion was performed with 50 ml of perfusion buffer through the portal vein of mice after anesthetizing. Livers were aseptically removed and minced in a sterile 10-cm cell culture dish with ice-cold perfusion buffer without collagenase. Then, hepatocytes were filtrated through a 70 µm cell strainer (BD Falcon) into a new tube followed with centrifugation at 50 g for 2 min at 4 °C. Cells were then washed with cold hepatocyte wash medium (Invitrogen) three times and re-suspended in 15 ml of cold HepatoZYME-SFM medium (Invitrogen) supplemented with 2 mM L -glutamine, 20 units/ml penicillin, and 20 μg/ml streptomycin. After trypan blue staining for determination of viability, cells were plated at 6 × 105 cells/well in 6-well culture dishes or at 3 × 105cells/well in 12-well dishes pre-coated with collagen. Cells were cultured for at least 8 h before further use. Cells were subsequently treated with the desired reagents prior to further analysis.
4
Isolation and Hypoxia Induction of Hepatic Cell Lines
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HepG2 cells (HB‐8065; ATCC, Manassas, VA, USA) were grown in Minimum Essential Medium (Invitrogen, USA), supplemented with 10% foetal bovine serum (Invitrogen), 100 mg/ml streptomycin, 100 IU/ml penicillin and 2 mM L‐glutamine. Primary hepatocytes were isolated and cultured from mice as previously described.27 Briefly, after mice were anesthetised, their livers were perfused via the portal vein with freshly prepared perfusion medium (Invitrogen), followed by digestion buffer (0.33 mg of collagenase I/ml). The livers were subsequently dispersed with hepatocyte wash medium (Invitrogen), and then seeded onto fibronectin‐coated plates (Corning, USA). For hypoxia induction in vitro, cells were incubated in an airtight chamber in an atmosphere of 1% O2, 94% N2 and 5% CO2 for the time periods indicated.
5
Isolation of Mouse Hepatocytes
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Mouse hepatocytes were isolated by a 2-step perfusion procedure.70 (link) Briefly, mice were anesthetized with ketamine (100 mg/kg), xylazine (10 mg/kg), and acepromazine (3 mg/kg). Following laparotomy, the liver was perfused in situ through the inferior vena cava with 20 mL of prewarmed liver perfusion medium (Invitrogen) followed by 40 mL of liver digestion medium (Invitrogen). The liver was then placed in ice-cold hepatocyte wash medium (Invitrogen), and the capsule of the liver was then gently disrupted in order to release the hepatocytes. The cell suspension was filtered with a 70-mm cell strainer (Becton, Dickinson), and cells were washed once (30′ g, 4 min, 4°C). Dead cells were removed using Percoll solution (Sigma-Aldrich) as described previously.71 (link) Live cells were washed, pelleted, resuspended in incubation medium, and seeded on 6-well Primaria plates (Becton, Dickinson) at a density of 4 ×105 cells/well. Cells were allowed to adhere to the plates for 3 h, after which the culture medium were replaced by fresh 2 mL medium.
6
Primary Hepatocyte Isolation by Perfusion
7
Isolation and Treatment of Primary Murine Hepatocytes
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Primary hepatocytes were isolated from mice using the collagenase type X (Wako, Japan; 039-17864) perfusion method.20 (link) Briefly, primary hepatocytes were isolated by collagenase type X (Wako Pure Chemical Industries, ltd, Japan; 039-17864) perfusion method. Cells were washed with hepatocyte wash medium (Thermo Fisher Scientific, Waltham, MA; 17704024), purified by Percoll (GE Healthcare, Chicago, IL; 17089101) density gradient separation, and resuspended in William’s E medium (Thermo Fisher Scientific; 12551032) with 5% fetal bovine serum, 10-nM dexamethasone, and 20-nM insulin. Cells were then seeded on collagen-coated plates at a final density of 3.5 × 104 cells/cm2. After 4 hours, attached cells were cultured with fresh medium and transduced with the indicated adenoviruses. Primary hepatocytes were treated for 4 hours with EBSS (Sigma-Aldrich, St. Louis, MO; E3024), containing 200-μM OA (16 hours; Sigma-Aldrich; O1008), 10-ng/mL TNF (16 hours; PeproTech), 1-μg/mL LPS (6 hours; Sigma-Aldrich; L2630), 50-μM SNAP (4 hours; Sigma-Aldrich, N3398), 100-nM PTIO (30 minutes; Sigma-Aldrich; P5084), and 20-μM CQ (Sigma-Aldrich, C6628). Insulin signaling in primary hepatocytes was stimulated with a 10-minutes exposure to 5-nM insulin (Sigma-Aldrich; I5500).
8
Isolation and Transplantation of Primary Murine Hepatocytes
9
Differentiation of HepaRG Cells for Hepatotoxicity Studies
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Human hepatic stem cell line HepaRG is the most suitable cell line for studying drug-induced hepatitis because of the richer CYP450 proteins in HepaRG than L-02, HepG2 and hiHeps cell lines 46 (link), 47 (link). HepaRG was obtained from ThermoFisher Scientific (#HPRGC10). The cells were seeded at 1 × 105 undifferentiated cells/cm2 in Hepatocyte Wash Medium (ThermoFisher Scientific, #17704024) containing additives for growth (Gibco). Then the cells were cultured at 37 °C with 21% O2 and 5% CO2 for 14 days before differentiation. The medium was renewed every 3 days. Cell differentiation was induced as described 48 (link). Briefly, the growth medium was composed of William's E medium supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, 5 μg/mL insulin, and 50 μM hydrocortisone hemisuccinate. For the routine differentiation process, a two-step procedure was used. Cells were first maintained in the growth medium for 2 weeks, then maintained in the differentiation medium (supplemented with 2% DMSO) for 2 more weeks. The cells were maintained up to 4 weeks after differentiation for use. As for the mouse macrophage RAW264.7 cells, they were cultured in DMEM-based complete medium.
10
Isolation and Characterization of Prostate Cancer-Associated Fibroblasts
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All tissue samples were acquired under an approved protocol by a University of Wisconsin-Madison Institutional Review Board. Biopsy punches (4 mm) were taken from normal and tumor areas of the prostate. A thin slice was taken from each punch to determine the presence of cancer, and the remaining tissue was mechanically minced and added to digestion buffer (0.5% collagenase; 0.1% Dispase; 1% PenStrep; 500 U/ml DNase 1 (Worthington Biochemical, Lakewood, NJ)) in Hepatocyte Wash Medium (Gibco, ThermoFisher, Waltham, MA) and incubated for 4 hours at 37°C. Cells were spun down and plated in FM (ScienCell, Carlsbad, CA) media at ~80 000 cells/well in a 24 well cell culture plate. Punches were confirmed to contain Normal/Tumor tissue by a certified pathologist using hematoxylin and eosin staining. Primary fibroblasts were analyzed by qPCR for expression of collagen and fibroblast activation protein (FAP) as markers of a cancer-associated phenotype (Supplementary Fig. S1 ).
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