Mouse primary hepatocytes were isolated according to a method described in 46 (link) with modifications. Briefly, under the perfusion, liver was washed in Ca2+ and Mg2+-free EBSS (Invitrogen) with 0.1 mM EGTA and 0.1 mM EDTA, and then digested with HBSS buffer (Invitrogen) containing 0.5 mg/ml collagenase IV and 0.5 mg/ml trypsin inhibitor (Roche). The perfused liver was dispersed and washed with DMEM. Hepatocytes were purified from the liver cells by gradient centrifugation in 50% Percoll (Invitrogen), and washed with DMEM. 1×106 hepatocytes were seeded onto each well of 6 well plates in DMEM supplemented with 10% FBS. William Medium E (Invitrogen) was used for the subsequent culture.
Trypsin inhibitor
Manufactured by Roche
Sourced in Switzerland
Trypsin inhibitor is a laboratory reagent used to inhibit the proteolytic activity of the enzyme trypsin. Trypsin is commonly used in cell culture procedures to detach adherent cells from the growth surface. Trypsin inhibitor can be used to stop this enzymatic activity when necessary.
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Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (12 mentions)
Collagenase a, by Roche (10 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Collagenase d, by Roche (9 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (7 mentions)
70 μm cell strainer, by Corning (6 mentions)
Papain, by Roche (5 mentions)
Trypsin edta, by Thermo Fisher Scientific (5 mentions)
Papain, by Merck Group (3 mentions)
Nerve growth factor (ngf), by Merck Group (3 mentions)
Dmem f 12 glutamax media, by Thermo Fisher Scientific (3 mentions)
24 well tissue culture plate, by BD (3 mentions)
B27 supplement, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (3 mentions)
Collagenase 4, by Roche (2 mentions)
Mg2 free ebss, by Thermo Fisher Scientific (2 mentions)
Hbss buffer, by Thermo Fisher Scientific (2 mentions)
Percoll, by Thermo Fisher Scientific (2 mentions)
William e medium, by Thermo Fisher Scientific (2 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions)
27 protocols using trypsin inhibitor
1
Isolation of Primary Mouse Hepatocytes
2
Isolation of Primary Mouse Hepatocytes
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Mouse primary hepatocytes were isolated according to a method described in 46 (link) with modifications. Briefly, under the perfusion, liver was washed in Ca2+ and Mg2+-free EBSS (Invitrogen) with 0.1 mM EGTA and 0.1 mM EDTA, and then digested with HBSS buffer (Invitrogen) containing 0.5 mg/ml collagenase IV and 0.5 mg/ml trypsin inhibitor (Roche). The perfused liver was dispersed and washed with DMEM. Hepatocytes were purified from the liver cells by gradient centrifugation in 50% Percoll (Invitrogen), and washed with DMEM. 1×106 hepatocytes were seeded onto each well of 6 well plates in DMEM supplemented with 10% FBS. William Medium E (Invitrogen) was used for the subsequent culture.
3
Cell Lysis and Protein Detection
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Cell were incubated for 10 minutes at 4°C in TNTE lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5% [v/v] Triton-X-100) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 μg/ ml pepstatin A (Sigma), 100 μg/ml benzamidine chloride (Calbiochem), and 1 mg/ml trypsin inhibitor, 10 μg/ ml antipain, 50 μg/ml aprotinin and 10 μg/ml leupeptin (Roche Applied Biosciences)), and phosphatase inhibitors (10 mM sodium pyrophosphate and 25 mM sodium fluoride (EM Sciences), and 1 mM sodium orthovanadate (Alfa Aesar)). Lysates were centrifuged at 15,000X g for 10 minutes at 4°C, and small aliquots were subjected to protein concentration determination using Bradford-based protein assays (Bio-Rad Laboratories). Cell extracts were resolved by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and were transferred onto nitrocellulose membrane (Bio-Rad Laboratory). The blots were incubated with mouse anti-FLAG (Sigma), rabbit anti-PIAS1 (Epitomics) or rabbit anti-actin (Sigma), as the primary antibody and HRP-conjugated donkey anti-mouse or anti-rabbit IgG (Jackson Laboratories) as secondary antibodies, followed by ECL and signal detection using a VersaDoc 5000 Imager (Bio-Rad Laboratories). Densitometry was performed using Quantity One software (Bio-Rad Laboratories).
4
Tongue Epithelium Fractionation and Protein Analysis
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Gastrointestinal tissue preparation was performed as described previously13 (link). Lingual epithelial tissue was exfoliated from the tongue by injection of an enzyme cocktail comprising 2.5 mg/mL dispase II (Wako Chemicals, Tokyo, Japan), 1.0 mg/mL collagenase D, and 1.0 mg/mL trypsin inhibitor (Roche Diagnostics, Basel, Switzerland) for 20 min at 25 °C, and then the epithelial tissue was peeled off. Proteins were separated by 7.5% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA) by electroblotting. Three subcellular fractions (cytoplasmic, membrane, and nuclear) of circumvallate papillae were prepared utilizing the subcellular protein fractionation kit (Thermo Fisher Scientific, Rockford, IL, USA) and characterized by western blotting using fraction-specific protein antibodies. The membranes were stained with rabbit anti-β-catenin (1:500; Cell Signaling Technology, Danvers, MA USA) and rabbit anti-β-actin (1:2000; Gene Tex, San Antonio, TX, USA) antibodies (see full-length blot strips in the Supplementary Information). Immunoreactivity was detected by enhanced chemiluminescence (Perkin Elmer Life Sciences, Inc.), and band density was determined using the FUSION solo5 (Vilber Lourmat, Marne-la-Vallée, France).
5
Primary Neural Cell Culture Protocol
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Dulbecco’s Modified Eagle’s Medium (DMEM), poly-D-lysine (PDL), putrescine, thyroxin, tri-iodothyroxine, progesterone, sodium selenite, papain, deoxyribonuclease l, bovine serum albumin (BSA) fraction V, and insulin were purchased from Sigma-Aldrich. L-glutamine, 0.25% trypsin-EDTA, and fetal bovine serum (FBS) were purchased from Invitrogen/Life Technologies. MEM was purchased from Gibco/Life Technologies. Holo-transferrin (human) was purchased from EMD Millipore. Trypsin inhibitor was purchased from Roche Life Science. FGF-basic and EGF were purchased from Peprotech. PDGF-AA was purchased from R&D Systems Inc.
6
Isolation and Culture of Primary Cells
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We purchased CFA, formalin, IL-1β, paraformaldehyde, DNAse I and Opti MEM culture medium from Sigma-Aldrich Company (St. Louis., MO). Collagenase, trypsin inhibitor and dispase-II were provided by Roche Diagnostics (Mannheim, Germany). B27 supplement was purchased from Invitrogen Company (Carlsbad, CA).
7
Isolation and Culture of GBM PDTCs
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We obtained GDC4032 (link),33 (link) which is an established GBM PDTC and GDC519 from freshly dissected samples from patients with GBM. GBM tumor tissues were mechanically minced using scalpels and scissors, then digested by shaking in 0.05% trypsin-EDTA (Thermo Fisher Scientific Inc.) at 37°C for 30–60 min. Trypsin inhibitor (Roche Diagnostics), DNase I (Thermo Fisher Scientific Inc.) and base medium (DMEM/F12 containing 15 mM HEPES [Wako Pure Chemical Industries]) and 1% Antibiotic-Antimycotic (Thermo Fisher Scientific Inc.) were added to the tissues and centrifuged at 180 × g for 5 min. Pelleted cells were incubated in cell culture flasks with serum-free culture medium at 37°C under a 5% CO2 atmosphere. Serum-free DMEM/Ham’s F12 culture medium (Wako Pure Chemical Industries) contained 20 ng/ml EGF (PeproTech), 20 ng/ml bFGF (PeproTech), 5 µg/ml heparin (Sigma-Aldrich Corp.), 10 ng/ml leukemia inhibitory factor (Merck Millipore), and 2% B27 supplements (Thermo Fisher Scientific Inc.). Human non-tumorous brain tissues were similarly processed as described above.
8
Isolation of Mouse Sensory Neurons
9
Dorsal Root Ganglia Neuron Isolation
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Cell culture was prepared as described previously45 (link). In brief, at postoperative days 5–7, control mice or CCD mice were anesthetized with Amobarbital Sodium (50 mg/kg ip), and the L4 and L5 DRGs were dissected out. The DRGs were placed in cold, oxygenated Complete Saline Solution (CSS), consisting of (in mM) 137 NaCl, 5.3 KCl, 1 MgCl2, 3 CaCl2, 25 Sorbitol and 10 HEPES (pH 7.2). For 20 min, the DRGs were digested with 0.35U/ml of Liberase TM (Roche, Manheim, Germany), then for 15 min with 0.25U/ml Librease TL (Roche, Manheim, Germany) and 30U/ml papain (Sigma, USA) in CSS containing 0.5 mM EDTA at 37 °C. The DRG neurons were suspended in DMEM medium containing 1 mg/mL trypsin inhibitor (Roche, Manheim, Germany) and 1 mg/mL bovine serum albumin (Sigma, USA) and then plated onto poly-D-lysine/laminin-coated glass coverslips (Bio-Coat; BD Biosciences, San Jose, CA). The DMEM medium had equivalent amounts of DMEM and F12 (Gibco, Grand Island, NK) with 10% FCS (Gibco, Auckland, New Zealand) and 1% penicillin and streptomycin (Invitrogen, Grand Island, NK). The cells were maintained in 5% CO2 at 37 °C in a humidified incubator and used within 16–24 hours after plating.
10
Dorsal Root Ganglia Neuron Isolation
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