HeLa human cervical cancer cells were purchased from the RIKEN BRC through the National Bio-Resource Project of the MEXT/AMED, Japan. The cells were cultured in DMEM, containing 10% fetal bovine serum and 1% Penicillin-Streptomycin Solution (×100) (FUJIFILM-Wako Pure Chemical Co., Osaka, Japan), at 37 °C under an atmosphere of 5% CO2. To each well of a 96-well microplate containing cell suspension (1 × 104 cells/mL, 200 µL) was added a sample dissolved in MeOH after a preincubation for 24 h, and the plate was incubated for 48 h. Then, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in PBS (1 mg/mL, 50 μL) was added to each well, and the plate was further incubated for 3 h. After removing the supernatant, the residue was dissolved in DMSO (150 μL). The absorbance at 538 nm was measured. Three replicates were examined to determine the IC50.
Penicillin streptomycin solution 100
Manufactured by Fujifilm
Sourced in Japan, United Kingdom
Penicillin-Streptomycin Solution (×100) is a sterile, concentrated liquid that contains the antibiotics penicillin and streptomycin. It is commonly used as a supplementary ingredient in cell culture media to prevent bacterial contamination.
Automatically generated - may contain errors
Lab products found in correlation
Knockout serum replacement (ksr), by Thermo Fisher Scientific (5 mentions)
Dmem high glucose, by Fujifilm (3 mentions)
Bone morphogenetic protein 4 truncated bmp 4, by Fujifilm (3 mentions)
Bfgf fgf2, by ReproCELL (2 mentions)
Fetal bovine serum (fbs), by GE Healthcare (2 mentions)
Mc3t3 e1, by Fujifilm (1 mentions)
Mc3t3 e1, by RIKEN BioResource Center (1 mentions)
α mem, by Fujifilm (1 mentions)
Recombinant human wnt3a protein, by R&D Systems (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Recombinant human mouse rat activin a protein, by R&D Systems (1 mentions)
Recombinant human hgf protein, by R&D Systems (1 mentions)
Dexamethasone (dex), by Fujifilm (1 mentions)
Recombinant human oncostatin m osm protein, by R&D Systems (1 mentions)
48 well culture plates, by BD (1 mentions)
Quantichrom calcium assay kit, by BioAssay Systems (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Biosera (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (1 mentions)
14 protocols using penicillin streptomycin solution 100
1
MTT Cytotoxicity Assay for HeLa Cells
2
Maintenance of HEK293T Cell Line
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HEK293T cells (obtained from Dr. Koji Shibasaki, Gunma University, Maebashi, Japan) were maintained in Dulbecco’s Eagle’s medium (DMEM high glucose; FUJIFILM Wako Pure Chemical Corporation) containing 10% fetal bovine serum (FBS; GE Healthcare, Buckinghamshire, UK) and penicillin–streptomycin solution (× 100) (FUJIFILM Wako Pure Chemical Corporation) at 37 °C and 5% CO2.
3
Culturing Murine Osteoblastic Progenitors
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The murine osteoblastic progenitor cell line MC3T3-E1 was obtained from the RIKEN Bioresource Center (RCB1126). MC3T3-E1 cells were maintained in minimum essential Eagle medium, alpha modification (α-MEM; FUJIFILM Wako Pure Chemical CorporationOsaka, Japan), supplemented with 10% fetal bovine serum (FBS; SERANA Brandenburg, Germany), a penicillin–streptomycin solution (×100) (FUJIFILM Wako Pure Chemical Corporation) at 37 °C, and 5% CO2.
4
Endoderm Differentiation of hiPSCs
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hiPSCs were induced to differentiate into endoderm (phase I) using 2mM L-Glutamine, 200 mM Solution (25030–081: ThermoFisher Scientific, Tokyo, Japan), 100 μM 2-mercaptoethanol (M3148-25ML:Sigma-Aldrich, St. Louis, MO), 1% Penicillin-Streptomycin Solution (×100) (16823191: FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 100 ng/mL Recombinant Human/Mouse/Rat Activin A Protein (338-AC-010: R&D Systems, Minneapolis, MN), 25 ng/mL Recombinant Human Wnt-3a Protein (5036-WN-010: R&D SYSTEMS, Minneapolis, MN), 0.29% Albumin, Human, recombinant expressed in plants (018–21541: FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) in RPMI 1640 with L-Gln, liquid medium (30264–85: Nacalai Tesque, Kyoto, Japan) for 24 h.
5
Directed Differentiation of hiPSCs into Endoderm
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hiPSCs were induced to differentiate into endoderm (phase II) using 1% Penicillin-Streptomycin Solution (×100) (16823191: FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 100 ng/mL Recombinant Human/Mouse/Rat Activin A Protein (338-AC-010: R&D Systems, Minneapolis, MN) in RPMI 1640 with L-Gln, liquid medium (30264–85: Nacalai Tesque, Kyoto, Japan), supplemented with 0.2% KSR (10828028: ThermoFisher Scientific, Tokyo, Japan) for 24 h.
6
Hepatocyte Induction from hiPSC-Derived Endoderm
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hiPSC-derived endoderm was further treated with 1% Penicillin-Streptomycin Solution (×100) (16823191: FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 20 ng/mL Bone Morphogenetic Protein 4 (truncated) (BMP-4) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 200 ng/mL Recombinant Human Sonic Hedgehog/Shh Protein, High Activity (8908-SH-005: R&D SYSTEMS, Minneapolis, MN), 10 ng/mL bFGF (FGF2) (ReproCELL Inc., Kanagawa, Japan) in RPMI 1640 with L-Gln, liquid medium (30264–85: Nacalai Tesque, Kyoto, Japan), supplemented with 2% KSR (10828028: ThermoFisher Scientific, Tokyo, Japan) for 5 days for the induction of hepatocytes.
7
Hepatic Maturation of hiPSC-Derived Endoderm
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hiPSC-derived endoderm was further treated with 1% Penicillin-Streptomycin Solution (×100) (16823191: FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 20 ng/mL Recombinant Human HGF Protein (294-HG-005: R&D SYSTEMS, Minneapolis, MN), 20 ng/mL Bone Morphogenetic Protein 4 (truncated) (BMP-4) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 10 ng/mL bFGF (FGF2) (ReproCELL Inc., Kanagawa, Japan) in RPMI 1640 with L-Gln, liquid medium (30264–85: Nacalai Tesque, Kyoto, Japan), supplemented with 2% KSR (10828028: ThermoFisher Scientific, Tokyo, Japan) for 5 days in initial stage of hepatic maturation.
8
Hepatic Maturation of hiPSC-derived Endoderm
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hiPSC-derived endoderm was further treated with 1% Penicillin-Streptomycin Solution (×100) (16823191: FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 10 ng/mL Recombinant Human Oncostatin M (OSM) Protein (295-OM-010: R&D Systems, Minneapolis, MN), 0.1 μM Dexamethasone (047–18863: FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) in RPMI 1640 with L-Gln, liquid medium (30264–85: Nacalai Tesque, Kyoto, Japan), supplemented with 2% KSR (10828028: ThermoFisher Scientific, Tokyo, Japan) for 6–16 days in the latter period of hepatic maturation.
9
Calcium Release and pH Measurements for WMTA
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The wells of 48-well culture plates (Becton Dickinson Labware, Lincoln Park, NJ, USA) were filled with 500 µl of α-MEM (Gibco-BRL, Grand Island, NY, USA) supplemented with 50 µg/ml streptomycin and 50 U/ml penicillin (Penicillin–Streptomycin Solution [×100], Wako, Osaka, Japan) containing 10% fetal bovine serum (Biosera; Nuaillé, France) (CM). Then, D-WMTA or Na-WMTA were placed on the bottoms of wells (one disc per well). Wells filled with CM without WMTA discs (No WMTA) were prepared as controls. All plates were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Media was collected on days 1, 7, 14, and 28 (n = 3 discs per time point). The amount of Ca2+ release was assessed with a QuantiChrom Calcium Assay Kit (Bio Assay Systems, Hayward, CA, USA), then measured in accordance with the manufacturer’s instructions using a microplate reader at an absorbance of 590 nm. The pH level was measured by Twin pH Meter II LQUA twin (Horiba Advanced Techno, Kyoto, Japan).
10
Cell Culture Protocol for Colon, Liver, and Colorectal Adenocarcinoma
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A human colon adenocarcinoma cell line SW480 (ECACC, N0. 87092801), liver hepatocellular cell line HepG2 (ECACC, No. 85011430) and epithelial colorectal adenocarcinoma cell line CaCO-2 (ECACC, No. 86010202) were grown at 37 °C with 5 % CO2 in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Wako, Osaka, Japan), supplemented with 10 % heat-inactivated fetal bovine serum (Invitrogen, Grand Island, NY) and 1 % Penicillin-Streptomycin Solution (×100) (Wako).
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