Primary hepatocytes were isolated as described previously (Li et al., 2016 (link); Ying et al., 2017 (link)). Briefly, mice were infused with a calcium free HEPES-phosphate buffer A (Calcium and magnesium-free PBS containing 0.2 μM EGTA, 10 mM HEPES, 1 mM glucose and 0.2% BSA, pH 7.4) via the vena cava for 3–5 min. After the color of the liver changed to a beige or light brown color, collagenase-containing buffer B (PBS with 1 mM magnesium and 1 mM calcium, 0.2% BSA, and 30 mM HEPES, 0.5mg/ml collagenase H) was perfused into liver. After the appearance of cracking on the surface of liver, perfusion was stopped immediately and the liver was excised into ice-cold buffer A. Cells from digested livers were teased out, suspended in Buffer A, filtered through 100 μm cell strainer, and centrifuged at 60 × g for 6 min at 4°C. The pellet was washed with Buffer B (no collagenase) twice and then mixed with Percoll (adjusted to physiological ionic strength with 10x PBS) to a final concentration of 36% and centrifuged at 100 × g for 10 min, 4°C. After removing the supernatant, the hepatocyte pellet was washed once with Buffer B (without collagenase) and then cultured in Williams Medium E containing 10% FBS on collagen-coated plates (GIBCO, Life Technologies) and antibiotics. After overnight incubation (16 hr), culture medium was refreshed.
Collagen coated plates
Manufactured by Thermo Fisher Scientific
Sourced in United States
Collagen-coated plates are a type of cell culture substrate that provide a surface for cell attachment and growth. The collagen coating mimics the natural extracellular matrix, promoting cell adhesion and supporting cellular functions. These plates are commonly used in a variety of cell culture applications.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Ham s f10, by Wisent (1 mentions)
Dulbecco s modified eagle medium, by Wisent (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Epilife medium, by Thermo Fisher Scientific (1 mentions)
Epilife defined growth supplement edgs, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Harvard Bioscience (1 mentions)
Tunicamycin, by Merck Group (1 mentions)
Penicillin streptomycin solution, by Merck Group (1 mentions)
Percol gradient, by Merck Group (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Collagenase d, by Thermo Fisher Scientific (1 mentions)
Collagenase d, by Roche (1 mentions)
Cell strainer, by BD (1 mentions)
Magnetic sorting, by Miltenyi Biotec (1 mentions)
Hep3b, by American Type Culture Collection (1 mentions)
Hepg2, by American Type Culture Collection (1 mentions)
Its supplement, by Merck Group (1 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
9 protocols using collagen coated plates
1
Isolation of Primary Murine Hepatocytes
2
Isolation and Differentiation of Primary Smn2B/- Myoblasts
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Smn2B/− primary myoblasts were isolated from hindlimb muscles of 3-week-old mice as originally described (27 (link)). Cells were cultured on collagen-coated plates (Gibco) and maintained under growth conditions using Ham's F10 (Wisent) media supplemented with 20% fetal bovine serum, 2.5 ng/ml human recombinant basic fibroblast factor (Invitrogen), and 2% penicillin/streptomycin (Gibco). Primary myoblasts seeded at equal density were induced to differentiate with Dulbecco’'s Modified Eagle Medium (Wisent) supplemented with 5% horse serum (Gibco) and 1% penicillin/streptomycin. To assess the impact of TSA on fusion, Smn2B/− myoblasts were treated with 100 nm of TSA for 24 h and then induced to differentiate for 72 h as previously described (19 (link)). In all experiments, cells were maintained at 37°C with 5% CO2 and were provided fresh media every other day.
3
Isolation of Primary Murine Hepatocytes
4
Cell Culture Maintenance Protocol
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All cells listed in Table S3 were cultured at 37°C with 5% CO2 in the indicated growth medium with regular passage every 2 to 3 days. Stable cell lines were maintained in culture medium with an appropriate concentration of puromycin (10 μg/ml for CHO-K1 cells and pgsA-745 cells and 2 μg/ml for other cell types). Primary human epidermal keratinocytes were cultured on collagen-coated plates (catalog number R011K; Gibco) and maintained in EpiLife medium (catalog number M-EPI-500-CA; Gibco) supplemented with EpiLife defined growth supplement (EDGS) (catalog number S-012-5; Gibco).
5
Motoneuron-like Cell Line ER Stress
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We used the NSC-34 motoneuron-like cell line cultured in Dulbecco’s modified Eagle’s medium high-glucose (DMEM, Biochrom, Berlin) supplemented with 10% fetal bovine serum and 1X penicillin/streptomycin solution (Sigma-Aldrich), on collagen-coated plates (Thermo-Fisher, Waltham, Massachusetts, USA) in a humidified incubator at 37 °C under 5% CO2. For ER stress we used added tunicamycin (0, 0.1, 1, or 10 µg/ml; Sigma-Aldrich) and MTT assay was performed as detailed in supplemenatry methods.
6
Isolation and Culture of Primary Hepatocytes
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Livers were perfused using liver perfusion buffer (HBSS KCl 0.4 g l−1, glucose 1 g l−1, NaHCO3 2.1 g l−1, EDTA 0.2 g l−1) and then digested using liver digest buffer (DMEM-GlutaMAX 1 g l−1 glucose, HEPES 15 mM pH 7.4, penicillin/streptomycin 1%, 5 mg per mouse Collagenase IV (C5138 Sigma)). After excision, livers were placed on ice in plating media (M199, foetal bovine serium 10%, penicillin/streptomycin 1%, sodium pyruvate 1%, l -glutamine 1%, 1 nm insulin, 1 mM dexamethasone, 2 mg ml−1 bovine serum albumin (BSA)). Tissue was homogenized using forceps and then filtered in plating media. Cells were then washed twice in plating media and then subjected to a 1:3 Percol Gradient (Sigma Aldrich). Cells were plated on collagen coated plates (Thermo Fisher Scientific) in plating media. Media was changed after 3 h to maintenance media (DMEM 4.5 g of glucose per l, penicillin/streptomycin 1%, l -glutamine 1%, 100 nM dexamethasone, 2 mg ml−1 BSA) for 12 h, and hepatocytes were treated as indicated.
7
Isolation of Primary Hepatocytes
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Primary hepatocytes were obtained by two-step perfusion with liver perfusion medium, SC-1 (137 mM NaCl, 5.4 mM KCl, 0.56 mM NaH2PO4⋅H2O, 0.85 mM Na2HPO4, 10 mM HEPES, 4.2 mM NaHCO3, 0.5 mM EGTA, 5 mM Glucose) followed by digestion medium, SC-2 (137 mM NaCl, 5.4 mM KCl, 0.56 mM NaH2PO4⋅H2O, 0.85 mM Na2HPO4, 10 mM HEPES, 4.2 mM NaHCO3 12 mM CaCl2⋅H2O) containing 0.5 mg/mL collagenase D (Roche, Indianapolis, IN). Liver cells were disaggregated by passing through a 100 μm pore nylon mesh Cell Strainer (BD Biosciences, San Jose, CA) and centrifuged at 100 × RCF for 10 min at 4 °C. Cells pellets were suspended in 36% percoll and centrifuged at 60 × RCF for 6 min. The subsequent cell pellets were washed with SC-2 buffer without collagenase-D, the numbers of total viable cells were determined by Trypan blue staining, and cells were plated on collagen-coated plates (Invitrogen, Carlsbad, CA). Primary hepatocyte cells were cultured in William E culture medium supplemented with 10% fetal bovine serum, and 100 U mL−1 of penicillin and 100 μg mL−1 of streptomycin at 37 °C. All animal studies were approved by the UCSD Institutional Animal Care and Use Committee.
8
Primary Mouse Hepatocyte Isolation and T3 Treatment
9
Isolation and Culture of Primary Human Hepatocytes and Immune Cells
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The hepatoma cell lines Hep3B, HepG2 were purchased from ATCC (Manassas VA) and propagated per recommendations. Huh 7 and Huh 7.5.1 were obtained from Dr. Jake Liang (NIH) and Dr. Francis Chisari (Scripps Research Institute), respectively, and propagated as previously described [31] (link). Primary human hepatocytes (PHH) were obtained from Dr. Andrew Cameron (Johns Hopkins). PHH were plated on collagen-coated plates (Invitrogen, Grand Island NY) and cultured in DMEM supplemented with 15 mM HEPES pH 7.5 (Cellgro, Manassas VA), 2 mM L-Glutamine (Cellgro), ITS supplement (Sigma-Aldrich, St. Louis MO) and 10 mg/mL gentamicin.
Freshly collected, de-identified human blood Leuko Paks were obtained from the Johns Hopkins Blood Donor and Therapeutic Center. PBMCs were isolated by Ficoll-Hypaque gradient centrifugation. T-cells, B-cells, monocytes, plasmacytoid and myeloid dendritic cells were isolated by magnetic sorting per the manufacturer's protocol (Miltenyi Biotec, Auburn CA) and cultured in RPMI 1640 media (Invitrogen, Grand Island NY), 10% heat-inactivated human AB serum (Gemini, West Sacramento CA) and 2 mM L-Glutamine. The purity of sorted subsets was determined by flow cytometry using lineage specific antibodies.
Freshly collected, de-identified human blood Leuko Paks were obtained from the Johns Hopkins Blood Donor and Therapeutic Center. PBMCs were isolated by Ficoll-Hypaque gradient centrifugation. T-cells, B-cells, monocytes, plasmacytoid and myeloid dendritic cells were isolated by magnetic sorting per the manufacturer's protocol (Miltenyi Biotec, Auburn CA) and cultured in RPMI 1640 media (Invitrogen, Grand Island NY), 10% heat-inactivated human AB serum (Gemini, West Sacramento CA) and 2 mM L-Glutamine. The purity of sorted subsets was determined by flow cytometry using lineage specific antibodies.
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