Primary human hepatocytes are thawed in Cryopreserved Hepatocyte Recovery Medium (CHRM, CM7000, ThermoFisher) and plated using Primary Hepatocyte Thawing/Plating supplements (CM3000, ThermoFisher), with a 1:40 dilution of Matrigel (354277, Corning) and at least 3% fetal bovine serum (FBS). 24 h post-plating, media is changed and Primary Hepatocyte Maintenence Supplements (CM4000, ThermoFisher), plus 1% FBS are added. All supplements are added to Williams E Media, with no phenol red (A1217601, ThermoFisher).
Cm7000
Manufactured by Thermo Fisher Scientific
The CM7000 is a centrifuge designed for laboratory applications. It provides reliable and consistent performance for a variety of sample processing needs. The CM7000 offers adjustable speed and time settings to accommodate different sample types and volumes.
Automatically generated - may contain errors
Lab products found in correlation
Cm4000, by Thermo Fisher Scientific (3 mentions)
Cm3000, by Thermo Fisher Scientific (3 mentions)
Phenol red, by Thermo Fisher Scientific (1 mentions)
William s e media, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Corning (1 mentions)
A5316, by Merck Group (1 mentions)
A32965, by Thermo Fisher Scientific (1 mentions)
William s medium, by Thermo Fisher Scientific (1 mentions)
Williams e medium, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Gemini Bio (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
4 protocols using cm7000
1
Thawing and Culturing Primary Human Hepatocytes
2
Investigating Hepatocyte Signaling Pathways
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Primary human, mouse, or rat hepatocytes were thawed in cryopreserved hepatocyte recovery medium (CM7000, Thermo Fisher Scientific) and plated in Williams E Media, with no phenol red (A1217601, Thermo Fisher Scientific) with primary hepatocyte thawing/plating supplements (CM3000, Thermo Fisher Scientific). Twenty-four hours postplating, the medium is changed and primary hepatocyte maintenance supplements (CM4000, Thermo Fisher Scientific) and 1% fetal bovine serum (FBS) are added. Hepatocytes were treated with recombinant hIL-6 (ab119444), mIL-6 (ab238300), rIL-6 (Cell Applications, RP3009), human HGF (R&D Systems, 294-HG-025), or mouse HGF (R&D Systems, 2207-HG-025) for 15 min at 50 ng/ml. Cells were lysed using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, 89900), containing protease and phosphatase inhibitors (Thermo Fisher Scientific, A32965 and 88667), run by SDS–polyacrylamide gel electrophoresis and probed using antibodies against pSTAT3 (Cell Signaling Technology, 9145, RRID:AB_2491009), total STAT3 (Cell Signaling Technology, 4904, RRID:AB_331269), pMET (Cell Signaling Technology, 3077, RRID:AB_2143884), and β-actin (Sigma-Aldrich, A5316, RRID:AB_476743).
3
Cryopreserved Hepatocyte Culturing Protocol
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Primary human hepatocytes (PHH) were purchased from Thermofisher scientific (HMCPMS; Thermofisher Scientific, MA, USA), and were cultured and maintained according to the supplemented protocol. In brief, the hepatocytes were thawed in Cryopreserved hepatocytes recovery medium (CM7000; Thermofisher Scientific), centrifuged at 100 x g for 10 min, and seeded at 2 × 106 cells/well on a collagen-coated plate in cryopreserved hepatocyte plating medium (CM9000; Thermofisher Scientific). After incubating the plate at 37 °C for 6 h, the culture medium was replaced by William’s medium (Gibco) supplemented with hepatocytes maintenance supplement pack (CM4000; Thermofisher Scientific).
4
Hepatocyte Culture and Apoptosis Induction
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Cryo-preserved primary human hepatocytes from single donors (PHH; BD Gentest, Tewksbury, MA; Lonza, Walkersville, MD; Thermo Fisher Scientific) and the human HCC cell line HuH7 (ATCC, Manassas, VA) were used for in vitro experiments. PHH were thawed according to the supplier's instructions using Cryopreserved Hepatocyte Recovery Medium (CM7000, Thermo Fisher Scientific), then resuspended in Williams Medium E (Sigma-Aldrich) with plating supplements (CM3000, Thermo Fisher Scientific) and plated onto collagen-coated PAA gels, glass, or 96-well plates at a density of 50,000 cells per cm 2 . HuH7 cells were cultured in DMEM (Corning) supplemented with 10% FBS (Gemini Bio-Products, West Sacramento, CA) and 1% penicillin-streptomycin (Corning) and seeded onto collagen-coated PAA gels or uncoated glass at a density of 18,750 cells per cm 2 . For positive control cells for apoptosis, PHH were treated with 5 µM staurosporine (STS) for 20 h one day after seeding.
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