Primary hepatocytes were isolated from 8-12-week-old C57BL6/J mice (20-25 g) using a two-step perfusion method, and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), as described previously (26 (link)). For pharmacological studies, cells were treated with 40 U/ml EPO, 0.3 mmol/l palmitate (PA; Sigma-Aldrich; Merck KGaA) or 1 µmol/l thapsigargin (Thp; Sigma-Aldrich; Merck KGaA) for 24 h. Where indicated, the cells were pre-treated with 40 µmol/l resveratrol (Sigma-Aldrich; Merck KGaA) or 2 µmol/l EX-527 (MedChem Express, LLC) for 1 h prior to EPO treatment. Primary hepatocytes isolated from EPO-treated and control mice were incubated with 1, 2 and 10 nmol/l human recombinant FGF21 (Abcam) for 4 h; vehicle treatment served as control.
Thapsigargin thp
Manufactured by Merck Group
Sourced in United States
Thapsigargin (Thp) is a lab equipment product used for its inhibitory effect on the Sarco/Endoplasmic Reticulum Ca2+ ATPase (SERCA) enzyme. It is a highly specific and potent inhibitor of SERCA, which plays a crucial role in calcium homeostasis within cells.
Automatically generated - may contain errors
Lab products found in correlation
Palmitate pa, by Merck Group (1 mentions)
Ex527, by MedChemExpress (1 mentions)
Resveratrol, by Merck Group (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
2 protocols using thapsigargin thp
1
Isolation and Culture of Primary Mouse Hepatocytes
2
Primary Hepatocyte Isolation and Culture
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Isolation and culture of C57BL/6J mice primary hepatocytes were previously described53 (link). HepG2 and FAO cell lines were grown in DMEM supplemented with 10% foetal bovine serum, 1% L-glutamine and 1% P/S (Invitrogen, NY, USA). The cells were sub-cultured in DMEM containing 0.2% bovine serum albumin (BSA) (Invitrogen, NY, USA) for 24 h until the cultures reached 70% to 80% confluence. The medium was replaced with DMEM containing BSA-conjugated OA/PA at concentrations of 0.5 mM and 1 mM, respectively, with or without BBR (5 μM), or 5% BSA as control for an additional 24 hours. Immortalised mouse normal hepatocytes (AML12) were cultured as previously described23 (link). PA, OA, TM, and thapsigargin (THP) were obtained from Sigma-Aldrich; insulin was provided by Novartis (NJ, USA).
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