Primary hepatocytes were isolated from 8- to 12-week-old male C57BL/6 mice by perfusion with liver digest medium (Invitrogen, 17703–034) followed by 70 μm mesh filtration. Percoll (Sigma, P7828) gradient centrifugation allowed primary hepatocytes isolation from other cell types and debris. Cells were seeded in plating medium (DMEM with 10% FBS, 2mM sodium pyruvate, 1% penicillin/streptomycin, 1μM dexamethasone, and 100nM insulin). After 4 h of seeding, the medium was changed and incubated in maintenance medium (DMEM with 0.2% BSA, 2mM sodium pyruvate, 1% penicillin/streptomycin, 0.1μM dexamethasone, and 1nM insulin). The following day (day 1) hepatocytes were treated overnight with the indicated compounds at 1μM. On day 2, media was changed to glucose production media (glucose free DMEM with 0.2% BSA, 20mM sodium pyruvate, 2mM sodium lactate, 1% penicillin/streptomycin, 4mM glutamine, sodium bicarbonate) supplemented with glucagon (200nM) and fresh compounds. After 4hrs of incubation, media was collected and glucose level was measured using a glucose assay kit from Eton Bioscience Inc.
Liver digest medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Liver Digest Medium is a cell culture medium designed for the isolation and cultivation of primary hepatocytes from liver tissue. It provides the necessary nutrients and growth factors to support the growth and maintenance of these specialized liver cells.
Automatically generated - may contain errors
Lab products found in correlation
Liver perfusion medium, by Thermo Fisher Scientific (42 mentions)
Percoll, by Merck Group (8 mentions)
William s e medium, by Thermo Fisher Scientific (7 mentions)
Percoll, by GE Healthcare (6 mentions)
Hepatocyte wash medium, by Thermo Fisher Scientific (6 mentions)
70 μm cell strainer, by BD (3 mentions)
Percoll solution, by GE Healthcare (3 mentions)
Wortmannin, by Merck Group (2 mentions)
Dynabeads magnetic beads, by BD (2 mentions)
Mouse lsec binding magnetic beads, by Miltenyi Biotec (2 mentions)
Hepatocyte wash buffer, by Thermo Fisher Scientific (2 mentions)
Percoll beads, by Merck Group (2 mentions)
Pre warmed liver perfusion medium, by Thermo Fisher Scientific (2 mentions)
L 15 medium, by Thermo Fisher Scientific (2 mentions)
Cell staining buffer, by BioLegend (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Cd8α t cell isolation kit, by Miltenyi Biotec (2 mentions)
Ls column, by Miltenyi Biotec (2 mentions)
Gentlemacs, by Miltenyi Biotec (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
74 protocols using liver digest medium
1
Isolation and Characterization of Primary Murine Hepatocytes
2
Generation of Humanized NRG Mice
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We constructed NRG-hu mice using a previously reported method [22 (link)]. Briefly, human CD34+ cells were isolated from 16- to 20-week-old fetal liver tissues (Advanced Bioscience Resources, Alameda, CA). The tissues were digested with liver digest medium (Invitrogen, Frederick, MD). The suspension was filtered through a 70-μm cell strainer (BD Falcon, Lincoln Park, NJ) and centrifuged for 5 min to isolate mononuclear cells by Ficoll gradient centrifugation. After selection with the CD34+ magnetic-activated cell sorting (MACS) kit, CD34+ hematopoietic stem cells were injected into the liver of each irradiated (300 rad) 2- to 6-day-old NRG mouse (0.5 × 106/mouse). More than 95% of the humanized mice were stably reconstituted with human leukocytes in the blood (60%–90% at 12–14 weeks). The level of engraftment was similar in each cohort. All the mice were housed at the University of North Carolina at Chapel Hill.
3
Isolation of Rat Primary Hepatocytes
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Primary hepatocytes were isolated from male Sprague–Dawley rats as described previously [24] (link). The portal vein and inferior vena cava of anesthetized animals were cannulated and perfused with 37 °C oxygenated perfusion media, pH 7.4, containing 118 mM NaCl, 5.9 mM KCl, 1.2 mM MgSO4, 1.2 mM NaH2PO4, 25 mM NaHCO3, 0.2 mM EGTA and 5 mM glucose. After 15 min, the liver was excised from the animal and perfused with liver digest medium (Invitrogen, Grand Island, NY). Then the cells were dispersed, washed four times, and suspended in attachment media, which consisted of 20 mM glucose DMEM supplemented with 30 mg/L proline, 100 mg/L ornithine, 0.544 mg/L ZnCl2, 0.75 mg/L ZnSO4·7H2O, 0.2 mg/L CuSO4·5H2O, 0.25 mg/L MnSO4, 2 g/L bovine serum albumin (Sigma), 5 nM insulin, 100 nM dexamethasone, 100,000 U penicillin, 100,000 U streptomycin, and 2 mM glutamine. After 4 h of incubation in the attachment media, the primary hepatocytes were switched to a maintenance media identical to the attachment media except it had a concentration of 1 nM (instead of 5 nM) insulin.
4
Isolation and Characterization of Mouse and Human Liver Sinusoidal Endothelial Cells
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Mouse LSECs were isolated by previously described two-step collagenase perfusion technique with modifications.15 (link) In brief, the liver was perfused with Liver Perfusion Medium (Invitrogen), and dissociated by Liver Digest Medium (Invitrogen). The NPCs were fractionated with Percoll (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) gradient centrifugation with 75% stock Percoll solution and 35% stock Percoll solution. LSEC faction was isolated by mouse LSEC-binding magnetic beads (Miltenyi, Auburn, CA, USA) and Dynabeads Magnetic Beads conjugated with anti-mouse CD31 antibody (MEC13.3, BD Biosciences). Expression of Id1, CXCR7, HGF and Wnt2 messenger RNA was determined. Primary human LSECs were procured from ScienCell Research Laboratories (catalog no. 5000, Carlsbad, CA, USA). Expression of factor VIII was validated by immunostaining. Akt-LSECs were derived from isolated LSECs that were transfected with the pCCL. PGK lentiviral vector with mouse constitutively active Akt1 (myristoylated Akt: myrAkt).63 (link) After starving in serum-free medium, 500 000 LSECs were seeded and stimulated with 10 ng ml−1 SDF-1. LSECs were also treated with 30 μm Wortmannin (Sigma-Aldrich).
5
Cell Culture and Infection Protocols
6
Isolation and Culture of Primary Hepatocytes
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Primary hepatocytes were cultured according to the published methods,43 (link) with a slight modification. Liver perfusion medium (Invitrogen, 17701-038) was used to perfuse the livers in situ via the portal vein and was followed by liver digest medium (Invitrogen, 17703-034) after anesthetizing the mice with pentobarbital sodium. The liver was excised and minced in William's E medium (Life technologies, A12176-01, Grand Island, NY, USA). The cell suspension was mixed gently several times with a pipette and strained through a steel mesh sieve after removing the liver capsule. The dispersed hepatocytes were collected via centrifugation at 50 × g for 5 min at 4 °C and washed twice with William's E medium. Hepatocytes were isolated by Percoll separation and washed twice with William's E medium. The final pellet was resuspended in William's E medium. The hepatocytes were counted, and their viability was determined by trypan blue exclusion. The hepatocytes were cultured under normoxic conditions (air/5% CO2) for further experiments.
7
Generation of Humanized Mouse Models
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We constructed humanized Balb/c rag2-γc (DKO) mice and Nod-rag1-γc (NRG) mice (The Jackson Laboratory) in a similar manner as previously reported [36 (link)]. Briefly, human CD34+ cells were isolated from 16- to 20-week-old fetal liver tissues (Advanced Bioscience Resources, Alameda, CA). Tissues were digested with liver digest medium (Invitrogen, Frederick, MD). The suspension was filtered through a 70-μm cell strainer (BD Falcon, Lincoln Park, NJ) and centrifuged at 150 × g for 5 minutes to isolate mononuclear cells by Ficoll. After selection with the CD34+ magnetic-activated cell sorting (MACS) kit, CD34+ HPCs (0.5 × 106) were injected into the liver of each 2- to 6-day-old DKO or NRG mice, which had been previously irradiated at 300 rad. More than 95% of the humanized mice were stably reconstituted with human leukocytes in the blood (60–90% at 12–14 weeks). Each cohort had similar levels of engraftment. All mice were housed at the University of North Carolina at Chapel Hill.
8
Isolation and Culture of Primary Mouse Hepatocytes
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Mouse primary hepatocytes were prepared as previously described (17 (link)). In short, mouse liver lobes were perfused with liver perfusion medium and liver digest medium (Gibco; Invitrogen) at 37°C. Dissociated cells from the tissue were separated using a 1.12, 1.08, and 1.06 g/ml Percoll (GE Healthcare) gradient. Hepatocytes were collected from the gradient solution and cultured in William’s E complete medium (Gibco; Invitrogen) in plates or glass coverslips coated with 0.2% gelatine. The viability of hepatocytes in each experiment preparation was assessed by vital staining with Trypan blue and by cell morphology in FACS analysis. All the experiments were performed with viability of hepatocytes preparations above 80% purity. The remaining dead cells were removed 12 h post-plating (12 h prior infection), as in freshly isolated preparations dead hepatocytes do not attach. HGF conditioning was performed by adding 100 ng/ml of recombinant mouse HGF (eBioscience) 1 h prior infection, with a subsequent wash before infection.
9
Isolation of Primary Mouse Hepatocytes
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Eight to ten-week-old mice were anesthetized by intraperitoneal injection of ketamine/xylazine (100mg/kg and 10mg/kg body weight). Following anesthesia, the abdominal cavity was opened by incisions with scissors and the vena cava and portal vein were located. A perfusion catheter was placed into the vena cava. Pre-warmed Liver Perfusion Medium (Invitrogen, 17701) (37C) was delivered at 1.6 ml/min for 12 min using a peristaltic pump. An incision was made at the portal vein as an outlet for the perfusion solution. Immediately following the Liver Perfusion Medium, pre-warmed Liver Digest Medium (Invitrogen, 17703) (37°C) was delivered at 1.6 ml/min for 12 min using a peristaltic pump. At the end of the perfusion, liver was dissected and transferred to a Petri dish on ice containing 10 ml of L-15 medium (Invitrogen, 21083) with 10%FBS. After washing three times with Hepatocyte Wash Buffer (Invitrogen, 17704), the primary hepatocytes were re-suspended in William’s E medium (Invitrogen, 12551) containing Percoll beads (Sigma, p4937). After 4 hrs incubation in William’s E medium containing 10%FBS penecillin and streptomycin, the unattached cells were removed, and the dishes were washed with PBS and incubated with maintenance media (William’s E medium containing 100µM Dex, Insulin-Transferrin-Selenium, 2mM glutamine and PenStrep).
10
Isolation of Lymphoid Cells from Liver
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The liver was perfused with liver perfusion medium (Invitrogen Life Technologies) followed by liver digest medium (Invitrogen Life Technologies) afterwards removed and digested for 30 min at 37 °C. Liver cells were gently pressed trough a cell strainer (∅ 70 µm) and lymphoid cells were separated by centrifugation 60 × g for 5 min. Lymphoid cells in the supernatant were collected, washed and resuspended in PBS supplemented with 1% FCS and diluted in 70% Easycoll (Biochrom) in a 1:1 ratio (≙ 35% Easycoll) and then overlaid onto 70% Easycoll and centrifuged for 20 min at 950 × g. Lymphoid cells were collected from the interface, washed and subsequently erythrocytes were lysed.
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