Embryos dissected from their dams at E7, E11, or E19 were cut into pieces with 0.05% collagenase IV solution (Invitrogen Life Technologies) and incubated in a shaking incubator for 1 h at 37 °C. The solution was filtered through a nylon cell strainer with 100 µm pore size (Corning Incorporated, Corning, NY, USA) and washed twice with Dulbecco's PBS (DPBS). The resultant cells were re-suspended in DMEM with high glucose (Hyclone) supplemented with 20% fetal bovine serum (FBS; Invitrogen Life Technologies) and 1% penicillin-streptomycin (Invitrogen Life Technologies), and then seeded into a 100 mm dish.
Collagenase 4 solution
Manufactured by Thermo Fisher Scientific
Collagenase IV solution is an enzyme preparation used to dissociate tissues and cells. It contains a mixture of enzymes that break down collagen, a primary structural component of the extracellular matrix.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Nylon cell strainer, by Corning (1 mentions)
Ko dmem, by Thermo Fisher Scientific (1 mentions)
Knockout serum, by Thermo Fisher Scientific (1 mentions)
Williams e medium, by Merck Group (1 mentions)
Percoll, by Merck Group (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Transferrin, by Merck Group (1 mentions)
Rat tail collagen 1, by Merck Group (1 mentions)
5 protocols using collagenase 4 solution
1
Embryonic Cell Isolation and Culture
2
Culturing Human Embryonic Stem Cell Lines
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The human embryonic stem cell lines, HUES1 and HUES9, having normal karyotype, were gifts from Dr. Douglas Melton (Harvard University), and were maintained according to the recommended culturing protocol. Briefly, cells were cultured on mitomycin-C treated mouse embryonic fibroblast (MEF) feeder cells in complete HUES medium consisting of 15% knockout serum replacement (Gibco-Invitrogen, Grand Island, NY, USA), 80% knockout Dulbecco modified Eagle medium (KO-DMEM, Invitrogen, Carlsbad, CA, USA), 1 mM L-glutamine, 0.1 mM beta-mercaptoethanol, 1% nonessential amino acids and 4 ng/mL human fibroblast growth factor [18] . For passage, cells were dissociated with the use of a collagenase IV solution (200 U/ml) (Gibco-Invitrogen). The cell culture was conducted in a 5% CO 2 air mixture at 37°C. The medium was changed daily (2ml/well). Karyotype analysis indicated normal karyotype in all experiments [47] .
3
hiPSC Embryoid Body Formation
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At 80% confluency, hiPSC colonies were passaged and dissociated into clumps with 0.1% collagenase IV solution (Thermo Fisher Scientific, Inc.). The cells were centrifuged (300 × g, 5 min, room temperature) in order to remove the collagenase and transferred into non-adherent 96-well plates (1,000 cells per well; Brand GmbH, Wertheim, Germany) in EB growth medium, which is a hiPSC growth medium without FGF-2. Embryoid bodies (EBs) formed within 24 h and were observed as free-floating aggregates. The culture medium was changed every 48 h. On day 7 the EBs were used for chondrogenic differentiation.
4
Hiinduced Pluripotent Stem Cell Differentiation
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At 80% confluency, hiPSC colonies were passaged and dissociated into clumps with 0.1% collagenase IV solution (Thermo Fisher Scientific, Inc.). The cells were centrifuged (300 × g, 5 min, room temperature) in order to remove the collagenase and transferred into non-adherent 96-well plates (1,000 cells per well; Brand GmbH, Wertheim, Germany) in EB growth medium, which is a hiPSC growth medium without FGF-2. EBs formed within 24 h and were observed as free-floating aggregates. The culture medium was changed every 48 h. On day 7 the EBs were used for chondrogenic differentiation.
5
Isolation and Culture of Primary Murine Hepatocytes
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The protocol of primary hepatocyte derivation was based on a published protocol [69 (link)]. Liver was obtained from 14-week-old C56BL/6 mice and digested using 2 mg/ml collagenase IV solution (Thermo Fisher Scientific) at 37 °C for 45 min. The digested tissue was plated in a 10-cm dish with Williams E medium (Sigma) supplemented with 5% FBS and mechanically dissociated. Then, cells were filtered using a 70-μm cell strainer, and 6 ml cell suspension was layered on top of a Percoll (Sigma) gradient of 1.12 g/ml, 1.08 g/ml, and 1.06 g/ml in PBS. Cells were centrifuged for 20 min at 800g and washed with Williams E medium with 5% FBS. After another centrifugation at 300g for 10 min, the cells were resuspended in Williams E medium with 5% FBS, 1% GlutaMAX, 50 U/ml Pen/Strep, 50 ng/ml EGF, 1 μg/ml insulin, 10 μg/ml transferrin (Sigma), and 1.3 μg/ml of hydrocortisone (Sigma) and plated on 10 μg/ml rat tail collagen I- (Sigma) coated plates with daily medium change.
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