For isolation of primary hepatocytes from mice, livers were perfused with collagenase type IV (Sigma-Aldrich), and hepatocytes were prepared as described previously 18 (link). The human hepatocarcinoma cell line HepG2 and murine hepatocyte cell line AML12 were obtained from the American Type Culture Collection (Manassas, VA, USA). HepG2 cells were maintained with Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. AML12 cells were maintained in DMEM/F12 (1:1) (Gibco, 11320-033, Co Dublin, Ireland) medium containing 10% FBS, 1% antibiotics, 1% ITSX (insulin, transferrin, selenium X) (Gibco, 51500-056), and 100 nM dexamethasone.
Aml12
Manufactured by Thermo Fisher Scientific
Sourced in United States
The AML12 is a laboratory instrument designed for the detection and analysis of various analytes. It utilizes advanced analytical techniques to provide reliable and accurate results. The core function of the AML12 is to facilitate the quantitative assessment of target compounds in a range of sample types. This equipment is intended for use in research and analytical laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Aml12, by American Type Culture Collection (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (4 mentions)
Hepg2, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Hepg2, by American Type Culture Collection (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Rnase free water, by Thermo Fisher Scientific (2 mentions)
Raw264, by American Type Culture Collection (2 mentions)
Dnase free water, by Thermo Fisher Scientific (2 mentions)
Raw 264, by Atlanta Biologicals (2 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (2 mentions)
16 protocols using aml12
1
Isolation and Culturing of Primary Hepatocytes
2
Hepatocyte Cell Culture and Treatments
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Mouse hepatocyte cell line, AML-12 (ATCC), was cultured in DMEM-F12 with 0.005 mg/mL insulin, 0.005 mg/mL transferrin, 5 ng/mL selenium and 40 ng/mL dexamethasone, 10% (v/v) fetal bovine serum (Gibco) and 1% (v/v) penicillin/streptomycin. Human hepatocellular carcinoma cell line, HepG2 (ATCC) was cultured in DMEM, 10% (v/v) fetal bovine serum (Gibco) and 1% (v/v) penicillin/streptomycin (Gibco). Both cell lines were grown at 37°C with 5% CO2. A working solution of C10 (100 µM) was prepared in 0.25% DMSO (Sigma-Aldrich). For palmitate and LPS treatments, cells (cell passages <20) were incubated with 750 µM palmitate (Sigma-Aldrich) solution conjugated to FFA-free BSA (Sigma-Aldrich), 2% in serum-free culture media or complete culture media containing 10 ng/mL LPS (Sigma-Aldrich).
3
Hypoxic AML12 Cell Stress Model
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Mouse hepatic parenchymal cell AML12 was purchased from ATCC. AML12 was cultured in DMEM/F12 with 10% fetal bovine serum (FBS; Gibco, USA) and 1% ITS (Insulin, Transferrin, Selenium, Cyagen, USA). AML12 cell were maintained at 37°C under stable 5% CO2 in a humidified chamber.
For hypoxia and reoxygenation modeling, AML12 was cultured in hypoxic incubator (94% N 2.5% CO2 and 1% O2) for 90 min and then stimulated with 100 ng/ml LPS (Sigma-Aldrich, USA) and 20 μM H2O2 in normal incubator.
For hypoxia and reoxygenation modeling, AML12 was cultured in hypoxic incubator (94% N 2.5% CO2 and 1% O2) for 90 min and then stimulated with 100 ng/ml LPS (Sigma-Aldrich, USA) and 20 μM H2O2 in normal incubator.
4
Culturing Human and Mouse Hepatocyte Cell Lines
5
Culturing AML-12 Mouse Liver Cells
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6
Palmitic Acid-Induced Senescence in Mouse Hepatocytes
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Mouse hepatocyte cell line (AML12) and mouse hepatic astrocyte line (JS1) were purchased from CAS Shanghai Cell Bank (Shanghai, China). AML12 cells were cultured in DMEM/F12 medium (containing 10% fetal bovine serum, Gibco, Grand Island, NY, USA), Insulin-Transferrin-Selenium supplement (Gibco), and 40 ng/mL dexamethasone (Sigma-Aldrich, Burligton, MA, USA). JS1 cells were cultured in DMEM/F12 medium (containing 10% fetal bovine serum) at 37 °C incubators containing 5% CO2.
AML12 cells were planted into the plate. Then cells were stimulated with palmitic acid (PA, 200 μM, Sigma-Aldrich, Burligton, MA, USA) for 48 h and collected for detection. AML12 cells were pre-treated with PTUPB for 1 h to observe the effect of PTUPB on PA-induced senescence of AML12 cells. An autophagy inhibitor 3-MA (5 mM, MCE, Dallas, TX, USA) or Sirt1 inhibitor EX527 (10 μM, Topscience, Shandong, China) was used to clarify the role of autophagy or Sirt1 in the anti-senescence effects of PTUPB in AML12 cells.
AML12 cells were planted into the plate. Then cells were stimulated with palmitic acid (PA, 200 μM, Sigma-Aldrich, Burligton, MA, USA) for 48 h and collected for detection. AML12 cells were pre-treated with PTUPB for 1 h to observe the effect of PTUPB on PA-induced senescence of AML12 cells. An autophagy inhibitor 3-MA (5 mM, MCE, Dallas, TX, USA) or Sirt1 inhibitor EX527 (10 μM, Topscience, Shandong, China) was used to clarify the role of autophagy or Sirt1 in the anti-senescence effects of PTUPB in AML12 cells.
7
Modulating ApoA4 Expression in Mouse Hepatocytes
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AML-12, a mouse hepatocyte cell line, was obtained from ShangCheng Beina ChuangLian Biology Technology Co., Ltd. (Shanghai, China). The cells were maintained under 37 °C and 5% CO2. The AML-12 cells were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (cat. 11320033, Gibco, New York, NY, USA)/Ham’s F-12 medium (cat. 11320082, Gibco, New York, NY, USA), with 40 ng/mL dexamethasone, 5 μg/mL insulin-transferrin-selenium (cat. I1884, Sigma-Aldrich, St. Louis, SL, USA), and 10% fetal bovine serum (cat. FB65011, CLARK Bioscience, Australia).
Before further extraction, cells were pre-treated with 200 μmol/L ethanol or left untreated (control) for 12 h. An APOA4-small interfering RNA (APOA4-si) kit and RiboFECT CP Transfection Kit were obtained from RiboBio (Guangzhou, China). The RiboFECT CP Transfection Kit was used to transfect APOA4-si into AML-12 cells to down-regulate APOA4 expression. After transfection for 36 h, the AML-12 cells were treated with EtOH for 12 h or left untreated. After 48 h of treatment, cells were harvested for subsequent experiments, such as cell viability and biochemical analyses. The sequences of the APOA4 siRNA were as follows:
Before further extraction, cells were pre-treated with 200 μmol/L ethanol or left untreated (control) for 12 h. An APOA4-small interfering RNA (APOA4-si) kit and RiboFECT CP Transfection Kit were obtained from RiboBio (Guangzhou, China). The RiboFECT CP Transfection Kit was used to transfect APOA4-si into AML-12 cells to down-regulate APOA4 expression. After transfection for 36 h, the AML-12 cells were treated with EtOH for 12 h or left untreated. After 48 h of treatment, cells were harvested for subsequent experiments, such as cell viability and biochemical analyses. The sequences of the APOA4 siRNA were as follows:
si-m-APOA4_001: 5′-GACCTGCAAGATCAGATCA-3′
si-m-APOA4_002: 5′-GCTGTAGAACAGTTTCAGA-3′
si-m-APOA4_003: 5′-GCAGCTGGAACAGTTCAGA-3′
8
Hepatocyte Cell Line Experiments
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Experiments were performed using normal hepatocyte cell line AML12 (ATCC®CRL-2254, Manassas, VA, USA), hepatoma cell lines Hepa 1-6 (ATCC®CRL-1830, VA, USA) and Hepa-1c1c7 (ATCC®CRL-2026, VA, USA). Cells were cultured in DMEM/F12 (Gibco, Waltham, MA, USA), 10% FBS (Gibco, Waltham, MA, USA), 2 mM L-glutamine, 1% penicillin and 1% streptomycin (10,000 U/mL, Gibco, MA, USA) at 37 °C and under 5% CO2. Cells were split when they reached 80% confluency. Cells were transfected with antisense oligonucleotides (ASOs) using Lipofectamine RNAiMAX (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol. Cells were analyzed 48 h after transfection. For insulin stimulation experiment AML12 cells were cultured for 2 h in reduced serum Opti-MEM media (Gibco, MA, USA) and then treated with 100 nM insulin solution (Paneco, Moscow, Russia).
9
Hypoxic Regulation of Liver Cell Crosstalk
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AML12 (alpha mouse liver 12 cells) and HSC-T6 (rat hepatic stellate cells) were purchased from the Shanghai Institute of Biochemistry and Cell Biology. Cells were cultured in DMEM-F12 (Hyclone, USA) supplemented with 10% heat-inactivated fetal bovine serum (Bioind, Israel), and were maintained in a humidified atmosphere with 5% CO2 at 37°C. For hypoxic exposure, AML12 cells were incubated in a tri-gas incubator (Thermo, America) of 1% O2, 5% CO2, and 94% N2. Serum-free “conditioned medium” (CM) was obtained from the supernatant of AML12 cells to eliminate the influence of serum cytokines. HSC-T6 cells were cultured in CM and the cells cultured in DMEM were used as control.
10
AML-12 Cell Culture and Experimental Protocols
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AML-12 (CRL-2254) cells were maintained at 37°C in DMEM-F12 1:1 containing 10% fetal bovine serum, 1x ITS (ThermoFisher Scientific, 41400), 10 nM dexamethasone and 1x penicillin/streptomycin in a 5% CO2 atmosphere. For siRNA transfection, cells were transfected using RNAiMAX (Thermo Fisher Scientific, 13778) with either specific siRNAs or negative siRNAs, and for overexpression experiments, Lipofectamine 3000 (Thermo Fisher Scientific, L3000001) with gene specific or empty plasmids, following the manufacturer's reverse-transfection protocol. Primary mouse hepatocytes were isolated and cultured using a standard 2-step collagenase perfusion protocol.80 (link) For knockdown studies, cells were treated with specific siRNA (10 nM) for 24-48 h before adding PA and GW3965. PA was prepared in the culture medium containing 2% BSA (SIGMA-ALDRICH, A4612) and added to cells for 12 or 24 h as indicated. The control cells were cultured in medium containing 2% BSA. Cells were cotreated with GW3965 and other drugs along with PA wherever mentioned.
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