CHO-K1, WIL2-S, AML-12, and MC38 cells were obtained from ATCC. CHO-K1 cells expressing either human PD-1 or PD-L1, aAPC/CHO-K1 cells (CS187110), and human PD-L1 positive aAPC/CHO-K1 cells (CS187108) were all brought from Shanghai Bangjing Industrial Co., Ltd (Shanghai, China). The XA-1 antibody gene sequences were cloned into the vector and expressed in CHO cells, then the proteins were purified via the protein A chromatography (Life Technologies). Human PD-1 (hPD-1) knock-in (C57BL/6 background) mice were brought from Shanghai Model Organisms Science and Technology Co., Ltd. (China, Shanghai). Human IL-2 and human IFN-γ ELISA kits were purchased from R&D System (USA). Recombinant PD-1, PDL1, and PD-L2 were obtained from R&D System (USA). Recombinant XA-1 purified from DNA plasmid or mRNA transfection in CHO or Expi293F cells, respectively, were performed according standard method for full length IgG antibody purification via protein A.
Aml12
Manufactured by American Type Culture Collection
Sourced in United States
The AML12 is a cell line derived from mouse liver cells. It is commonly used in cell culture and experimental research applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (15 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (12 mentions)
Hepg2, by American Type Culture Collection (11 mentions)
Dexamethasone (dex), by Merck Group (10 mentions)
Streptomycin, by Thermo Fisher Scientific (10 mentions)
Penicillin, by Thermo Fisher Scientific (9 mentions)
Aml12, by Thermo Fisher Scientific (8 mentions)
Hepa1 6, by American Type Culture Collection (7 mentions)
Raw264, by American Type Culture Collection (6 mentions)
Transferrin, by Thermo Fisher Scientific (5 mentions)
Aml 12, by Merck Group (4 mentions)
Hepg2, by Thermo Fisher Scientific (4 mentions)
Insulin, by Thermo Fisher Scientific (4 mentions)
Co2 incubator, by Thermo Fisher Scientific (4 mentions)
Hek293t, by American Type Culture Collection (3 mentions)
Mcf 10a, by American Type Culture Collection (3 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (3 mentions)
72 protocols using aml12
1
Characterization of XA-1 Antibody Targeting PD-1/PD-L1
2
Alcohol-Induced Hepatocyte Dysfunction
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AML-12 cells were grown in DMEM-F12 medium (Corning) containing a cocktail of insulin, selenium, and transferrin (41400-045 Thermo Scientific), 0.05 μg/mL dexamethasone (D4902 Sigma), 10% serum and antibiotics. Cells were treated with ethanol (E7023 Sigma) 100 mM (twice a day) for 48 h. AML-12 cells were treated with MG132 10 μM and chloroquine (CQ) 50 μM 6 h and 16 h before harvesting, respectively. HepG2 cells were grown in DMEM medium (Corning) containing 10% serum and antibiotics. After the incubation period, RNA or protein was isolated for various assays described below. AML-12 (Cat.: CRL-2254) and HepG2 (HB-8065) cells were purchased from ATCC. We used HepG2 cells in two figures, to show PIN1 expression alters the mitochondrial content of MATα1 in a human liver cell line in Fig. 3 and that S114A MAT1A mutant interacts much less with PIN1 in Fig. 4 . This cell line was authenticated in 2014 by STR testing and no anomalies were detected. We have used this cell line extensively because of ease of transfection and more importantly because it expresses MAT1A.
3
Oxidative Stress Response in Liver Cells
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miR-706 mimic and scrambled oligonucleotides were purchased from Oligobio (China) and transfected into L-02 and AML12 cell lines (ATCC, Manassas, VA) using Lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer’s instructions. siRNA oligonucleotides (Oligobio, China) were used to target the PKCα and TAOK1 transcripts (Table 1 ). After 8 h of transfection, cells were treated with 300 μM H2O2 for 1 h and 24 h harvested for mRNA analysis of bax, bad, bak, and bbc3, or for 48 h and harvested for protein analysis of α-SMA, PKCα, and TAOK1.
4
Maintenance and Knockdown of AML-12 Cells
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AML-12 (CRL-2254) cells were purchased from ATCC and maintained at 37 °C in DMEM/F12 1:1 containing 10% FBS, 1x ITS, 10 nM dexamethasone and 1x penicillin/streptomycin in a 5% CO2 atmosphere. Cells were placed in DMEM/F12 1:1 without serum or supplements for 24 h prior to passaging or knock-down. For all siRNA experiments, cells were transfected using RNAiMAX following the manufacturer’s protocol for reverse-transfection. Unless otherwise noted, assays were performed 96 hrs. after siRNA transfection. Knock-down efficiency was ascertained by western blotting for G6PC, a typical knock-down result is shown in Suppl. Fig. 1 .
5
Luciferase Assay for Cell-Based Transfection Experiments
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The human hepatocyte cell line HC-04 (provided by José Manautou, University of Connecticut) was maintained in equal volumes of DMEM and Ham’s F-12 media (Gibco) supplemented with 10% FBS (Gibco). HEK293T (CRL-3216), AML12 (CRL-2254) or Hepa 1 (CRL-1830) cells (ATCC) were maintained in DMEM with 10% FBS, 100 IU/mL penicillin G, and 100 μg/mL streptomycin (Invitrogen). They were incubated in a 37°C in a humidified incubator with a 5% CO2 atmosphere. Transfection experiments were performed using X-tremeGENE HP DNA transfection reagent (Roche). The luciferase reporter assay was performed using Dual-Glo Luciferase Assay System, as described previously (18 (link)). The luciferase activity was normalized to Renilla luciferase activities in the same sample.
6
Culturing Mouse Hepatocyte Cell Lines
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Mouse hepatocellular carcinoma cells (Hepa 1-6) and mouse immortalized hepatocytes (AML12) were purchased from ATCC (Manassas, VA). Hepa 1-6 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Thermo Fisher Scientific, Waltham, MA) containing 4mM L-glutamine, 4.5g/L glucose, 1mM sodium pyruvate, and 1.5g/L sodium bicarbonate, supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin as recommended by ATCC. AML12 cells were cultured in DMEM: F-12 Medium containing 2.5mM L-glutamine, 15mM HEPES, 0.5mM sodium pyruvate, and 1200mg/L sodium bicarbonate, supplemented with 10% FBS, 1% penicillin/streptomycin, 10μg/ml insulin, 5.5μg/ml transferrin, 5ng/ml selenium, and 40ng/ml dexamethasone.
7
Cell Culture Protocols for HEK-293T and AML12
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8
Hepatoprotective Effects of 2-Hydroxybutyrate
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Male SD and Wistar rats (160–180 g) and C57 mice (18–22 g) were purchased from Shanghai SLAC Laboratory Animal Company and housed at constant temperature (24 ± 2 °C) with a 12 h light-dark cycle. BRL3A (ATCC number: CRL-1442) and AML12 (ATCC number: CRL-2254) cells were purchased from ATCC (Manassas, MA, USA). Vac was purchased from Lilly Company (USA). AP, 2-HB, phenacetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium (±)-2-hydroxybutyrate-2,3,3-d3 (d3-2-HB) was purchased from C/D/N Isotopes Inc (Canada). ALT, AST kits were purchased from Nanjing Jiancheng Bioengineering Institute (China). GSH & GSSG and CCK-8 kits were purchased from Beyotime Biotechnology (China). Gut bacterial DNA extraction kit was purchased from QIAGEN Company (Germany). TRIzol reagent, reverse transcript enzyme and SYBR Green master mix were purchased from Invitrogen (Carlsbad, CA). CYP2E1 (Cat: ab28146, Lot:GR324351-11), CYP3A4 (Cat: ab3527, Lot: GR3210692-3), NQO-1 (Cat: ab2346, Lot: GR3550-44) primary antibodies were purchased from Abcam (USA). Collagenase type I (Cat: LS004196) was purchased from Washington Biochemical Corporation. Percoll (Cat: P1644) was purchased from Sigma-Aldrich.
9
Huh7 and Hepatoma Cell Culture
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Huh7 (JCRB0403) cells were purchased from JCRB, 293 T (CRL-3216) and Hepa1-6 (CRL-1830), SNU-499 (CRL-2234), PLC (CRL-8024), C3A (CRL-10741), Hepa-1c1c7 (CRL-2026), BNL (CRL-3308), AML12 (CRL-2254) cells were purchased from ATCC. Huh7, Hepa 1-6, Hepa-1c1c7, BNL lines were cultured in DMEM with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 2 mM sodium pyruvate; additional 22 mM of HEPES buffer is added to 293 T cells. SNU-499 were cultured in RPMI-1640 medium. PLC, C3A were culture in EMEM with 10% FBS. AML12 were cultured in DMEM-F-12 with 10% FBS. Cells were seeded and transduced after 24 hrs with a MOI (multiplicity of infection) of 1e4 vg/cell, and incubated for another 48 hrs for most of experiments, unless indicated for 24 hrs.
10
Characterization of Mouse and Human Kidney Cell Lines
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The mouse hepatocytes cell line AML-12 (alpha mouse liver 12) was obtained from ATCC and grown at 37°C in a humidified atmosphere of 5% CO2/95% air in Dulbecco’s Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F-12) supplemented with 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 5 ng/ml selenium, 40 ng/ml dexamethasone, 0.5 mM tryptophan and 1% gentamycin.
Proximal tubular cell line HK-2 (human kidney 2) was purchased from ATCC and grown at 37°C in a humidified atmosphere of 5% CO2/95% air in normal DMEM medium (Gibco) including 10% FCS (Gibco), 10 units per ml penicillin, 0.5 mM tryptophan and HEPES for buffering. ACMSD inhibitor was initially diluted from powder in DMSO until the stock concentration of 1 mM. This stock was further diluted with water until the 100 μM solution, which was used for the cell treatments.
All the cell lines purchased from ATCC have been authenticated by morphology, karyotyping and PCR-based approaches. All the used cell lines have been routinely checked in the laboratory for mycoplasma contamination with the MycoProbe detection kit (R&D systems). Only cells negative for mycoplasma contamination were used.
Proximal tubular cell line HK-2 (human kidney 2) was purchased from ATCC and grown at 37°C in a humidified atmosphere of 5% CO2/95% air in normal DMEM medium (Gibco) including 10% FCS (Gibco), 10 units per ml penicillin, 0.5 mM tryptophan and HEPES for buffering. ACMSD inhibitor was initially diluted from powder in DMSO until the stock concentration of 1 mM. This stock was further diluted with water until the 100 μM solution, which was used for the cell treatments.
All the cell lines purchased from ATCC have been authenticated by morphology, karyotyping and PCR-based approaches. All the used cell lines have been routinely checked in the laboratory for mycoplasma contamination with the MycoProbe detection kit (R&D systems). Only cells negative for mycoplasma contamination were used.
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