In this study, HEK 293-hERG cells (Merck Millipore, Billerica, MA, USA), which are HEK 293 cells stably expressing hERG, and CHO-hERG cells (Merck Millipore), which are CHO cells stably expressing hERG, were used. The HEK 293 cells were cultured in an equal volume mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Nutrient Mixture F-12 (Invitrogen Life Technologies, CA, USA) supplemented with 10 % fetal bovine serum (FBS) (Invitrogen Life Technologies) and 1 % penicillin/streptomycin (Invitrogen Life Technologies), and the CHO cells were cultured in Ham’s F12 medium (Invitrogen Life Technologies) supplemented with 10 % FBS and 1 % penicillin/streptomycin in a humidified 5 % CO2 atmosphere at 37 °C. For patch-clamp experiments, cultured cells on a polystyrene culture dish (Sumitomo Bakelite, Tokyo, Japan) were detached by Accutase (Innovative Cell Technologies, Inc., CA, USA) at room temperature for several minutes. A viable single cell suspension was obtained by resuspension in patch clamp solution with mild pipetting to avoid cell damage.
Nutrient mixture f 12
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, China
Nutrient Mixture F-12 is a cell culture medium formulation developed for the growth and maintenance of various cell types, including mammalian cells. It provides a balanced combination of essential nutrients, vitamins, and other components required for cell proliferation and survival.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (29 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (19 mentions)
Streptomycin, by Thermo Fisher Scientific (10 mentions)
Penicillin, by Thermo Fisher Scientific (10 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
Hydrocortisone, by Merck Group (6 mentions)
Trypsin edta, by Thermo Fisher Scientific (5 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Merck Group (4 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Arpe 19, by American Type Culture Collection (3 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (3 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Sartorius (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Cholera toxin, by Merck Group (3 mentions)
92 protocols using nutrient mixture f 12
1
Patch-clamp Methodology for hERG-expressing Cells
2
ANEPPS Staining of HEK293 Cells
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HEK293 cells (American Type Culture Collection) were maintained in 1:1 Dulbecco’s modified Eagle medium and nutrient mixture F-12 (Invitrogen) supplemented with 10% fetal bovine serum (Sigma-Aldrich), geneticin (0.6 mg/ml; G418, Life Technologies), and puromycin (5 μg/ml; Life Technologies). Cells were grown on 35-mm glass-bottom dishes until they reached 90% confluency. The same protocol was applied to self-spiking HEK293 cells. For ANEPPS staining, ANEPPS solution in DMSO was added directly to the cells in a 35-mm glass-bottom dish to a final concentration of 0.1 μM. Cells were then incubated at 4°C for 5 min before imaging.
3
Human Bone Marrow Mesenchymal Stem Cell Isolation
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Human bone marrow derived mesenchymal stem cells (MSCs) were purchased from Lonza (Morristown, NJ, USA). Dulbecc’s modified Eagl’s medium (DMEM), Nutrient Mixture F-12 (HAM), fetal bovine serum (FBS), antibiotics and trypsin-EDTA were procured from GIBCO Invitrogen (Carlsbad, CA, USA). CellTiter 96® Aqueous one solution was obtained from Promega (Madison, Wisconsin, USA). Hexafluoro-isopropanol (HFIP), Alizarin Red S and cetylpyridinium chloride, were purchased from Sigma-Aldrich, Singapore.
4
Isolation of Primary Microglia from Neonatal Mice
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Primary microglia cells were isolated from 2-day-old ICR mice as described previously [29 (link)]. In brief, whole brains of neonatal mice were taken, blood vessel and meninges were carefully removed. Then, the whole brains of 12 mice were polled together and finely minced. Next, incubated with 0.25% trypsin-EDTA solution (0.25% trypsin and 1 mM EDTA; Invitrogen) at 37°C for 15 min. The enzymatic reaction was quenched by the addition of 20% FBS. After centrifugation at 200 × g for 10 min. at room temperature, the pellet was resuspended in the DMEM: Nutrient Mixture F-12 (Invitrogen) supplemented with 10% FBS. After 2 weeks, microglia cells were harvested by mild shaking of the flask and collected by centrifugation. Purity of microglia was analysed by IBA-1(1:3000; Wako, Osaka, Japan) cytohistochemistry.
5
Infection of AGS Cells by H. pylori
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H. pylori strains were grown on gas chromatography (GC) agar plates (Oxoid, Wesel, Germany) supplemented with horse serum (5%), vancomycin (10 μg/ml), trimethoprim (5 μg/ml), and nystatin (1 μg/ml) (serum plates) and incubated for 2–3 days under microaerobic conditions (85% N2, 10% CO2, 5% O2) at 37 °C. Campylobacter jejuni C64 [30 (link)] was grown on Columbia blood agar plates (Oxoid) under microaerobic conditions.
Human gastric adenocarcinoma AGS (ATCC CRL 1739) were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in RPMI 1640 (Invitrogen, Germany) supplemented with horse serum (10%) under standard conditions. Cells at 70% confluence were starved for 12 h in Nutrient Mixture F12 (Invitrogen) and then infected with a multiplicity of infection (MOI) of 100 for 5 h. The cell culture supernatants were preserved at –70 °C for quantification of IL-8.
Human gastric adenocarcinoma AGS (ATCC CRL 1739) were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in RPMI 1640 (Invitrogen, Germany) supplemented with horse serum (10%) under standard conditions. Cells at 70% confluence were starved for 12 h in Nutrient Mixture F12 (Invitrogen) and then infected with a multiplicity of infection (MOI) of 100 for 5 h. The cell culture supernatants were preserved at –70 °C for quantification of IL-8.
6
ARPE-19 Cells Pretreatment and UPM Exposure
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The human RPE cell line ARPE-19 was obtained from the American Type Culture Collection (Manassas, MD, USA). The cells were maintained in Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (Invitrogen-Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum at 37 °C in a 5% CO2 incubator. When the cells were approximately 80% confluent, the cells were pretreated with or without 5 mM N-acetylcysteine (NAC, Sigma-Aldrich Chemical Co., St. Louis, MO, USA) or 200 μM necrostatin-1 (Sigma-Aldrich Chemical Co.). After 1 h, the cells were incubated with a desired concentration of UPM for 24 h.
7
Biomimetic Scaffold for Tissue Engineering
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Polycaprolactone (PCL), hyaluronic acid, (HA) minocycline hydrochloride (MH), 1,1,1,3,3,3-hexafluoro-isopropanol (HFIP), methanol, Alizarin red-S, cetylpyridinium chloride were purchased from Sigma-Aldrich. Silk fibroin (SF) was purchased from Zhang Peng International Trading, Singapore. Dulbecco’s modified eagle’s medium (DMEM), nutrient mixture F-12, fetal bovine serum (FBS), antibiotics and trypsin-ethylene diamine tetra acetic acid (EDTA) were procured from GIBCO (Invitrogen, Carlsbad, CA, USA). CellTiter 96® Aqueous one solution was obtained from Promega, Madison, WI, USA.
8
Primary Culture of Rat Dorsal Root Ganglia
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Poly-L-Lysine (P1399, Sigma-Aldrich), Laminin (L2020, Sigma-Aldrich), calcium/magnesium free Hank's Balanced Salt Solution (HBSS) (14170, Life technologies), Trypsin (T5266, Sigma-Aldrich, pH = 7.2), DNAse
9
Cultivation of Human Retinal Pigment Epithelial Cells
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The human retinal pigment epithelium cell line ARPE-19 (passage 19) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and nutrient mixture F-12 (Life Technologies, Carlsbad, CA, USA). The medium was supplemented with 10% HyClone fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL of penicillin, 100 μg/mL of streptomycin (Lonza, Basel, Switzerland or Life Technologies, Carlsbad, CA, USA for both), and 2 mM of L-glutamine (Life Technologies, Carlsbad, CA, USA). The cells were passaged every 3–4 days using 0.25% Trypsin–EDTA (Life Technologies, Carlsbad, CA, USA) and used for experiments in passages between 26 and 35. The cells were maintained routinely and during experiments in an incubator providing a humidified atmosphere at +37 °C with 5% CO2.
10
NGF-stimulated pERK Activation Assay
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Neuroscreen-1 PC12 cells (Thermo Scientific) were plated at 75,000 cells per well on PDL-coated Greiner 96-well tissue culture plates in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (Life Technologies). Cells were stimulated by the addition of recombinant human β-NGF (R&D Systems, 5 ng/mL final assay concentration), which had been pre-mixed with anti-NGF or isotype control antibodies. 15 min after addition of the NGF, cell supernatants were rapidly removed and cells lysed in 50 μL of lysis buffer from Phospho-ERK Cellular HTRF Assay Kit (Cisbio, Codolet, France). pERK HTRF assay was run according to manufacturer’s instructions.
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