Structured Review

Nacalai hepes
Effects of pH and temperature on the kinase activity of Pcal_0041. (A) Effect of pH. Kinase activity of Pcal_0041 was examined at 85°C in various buffers: ■, <t>MES-NaOH</t> (pH 5.5 to 7.0); □, <t>HEPES-NaOH</t> (pH 7.0 to 8.0); ●, Tricine-NaOH (pH 8.0 to 9.0); ○, CHES-NaOH (9.0 to 10); ▲, CAPS-NaOH (10.0 to 11.0). (B) Effect of temperature. Enzyme activity was examined in HEPES-NaOH buffer (pH 8.0) at various temperatures from 50 to 90°C. (C) Thermostability of Pcal_0041 at 100°C. Pcal_0041 was heated at 100°C for various intervals of time and the residual activity was examined at 85°C and pH 8.0. All buffers were prepared at the temperatures of use. Each point is the average from at least three measurements. SD values are indicated with bars. (D) Circular dichroism studies on recombinant Pcal_0041 protein. CD spectra from 190 to 280 nm were measured at various temperatures: ◆, 30°C; ■, 50°C; ▲, 70°C; ✖, 80°C; ○, 90°C.
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1) Product Images from "A Phosphofructokinase Homolog from Pyrobaculum calidifontis Displays Kinase Activity towards Pyrimidine Nucleosides and Ribose 1-Phosphate"

Article Title: A Phosphofructokinase Homolog from Pyrobaculum calidifontis Displays Kinase Activity towards Pyrimidine Nucleosides and Ribose 1-Phosphate

Journal: Journal of Bacteriology

doi: 10.1128/JB.00284-18

Effects of pH and temperature on the kinase activity of Pcal_0041. (A) Effect of pH. Kinase activity of Pcal_0041 was examined at 85°C in various buffers: ■, MES-NaOH (pH 5.5 to 7.0); □, HEPES-NaOH (pH 7.0 to 8.0); ●, Tricine-NaOH (pH 8.0 to 9.0); ○, CHES-NaOH (9.0 to 10); ▲, CAPS-NaOH (10.0 to 11.0). (B) Effect of temperature. Enzyme activity was examined in HEPES-NaOH buffer (pH 8.0) at various temperatures from 50 to 90°C. (C) Thermostability of Pcal_0041 at 100°C. Pcal_0041 was heated at 100°C for various intervals of time and the residual activity was examined at 85°C and pH 8.0. All buffers were prepared at the temperatures of use. Each point is the average from at least three measurements. SD values are indicated with bars. (D) Circular dichroism studies on recombinant Pcal_0041 protein. CD spectra from 190 to 280 nm were measured at various temperatures: ◆, 30°C; ■, 50°C; ▲, 70°C; ✖, 80°C; ○, 90°C.
Figure Legend Snippet: Effects of pH and temperature on the kinase activity of Pcal_0041. (A) Effect of pH. Kinase activity of Pcal_0041 was examined at 85°C in various buffers: ■, MES-NaOH (pH 5.5 to 7.0); □, HEPES-NaOH (pH 7.0 to 8.0); ●, Tricine-NaOH (pH 8.0 to 9.0); ○, CHES-NaOH (9.0 to 10); ▲, CAPS-NaOH (10.0 to 11.0). (B) Effect of temperature. Enzyme activity was examined in HEPES-NaOH buffer (pH 8.0) at various temperatures from 50 to 90°C. (C) Thermostability of Pcal_0041 at 100°C. Pcal_0041 was heated at 100°C for various intervals of time and the residual activity was examined at 85°C and pH 8.0. All buffers were prepared at the temperatures of use. Each point is the average from at least three measurements. SD values are indicated with bars. (D) Circular dichroism studies on recombinant Pcal_0041 protein. CD spectra from 190 to 280 nm were measured at various temperatures: ◆, 30°C; ■, 50°C; ▲, 70°C; ✖, 80°C; ○, 90°C.

Techniques Used: Activity Assay, Recombinant

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Centrifugation:

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Cell Cycle Assay:

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Construct:

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SYBR Green Assay:

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Incubation:

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Stripping Membranes:

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Infection:

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Expressing:

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Modification:

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Transfection:

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Article Snippet: .. The transfected cells were lysed in 250 µl HMDEKN cell-lysis buffer (10 mM Hepes, pH 7.4, 5 mM MgSO4 , 1 mM DTT, 0.5 mM EDTA, 25 mM KCl, and 0.5% NP-40) containing protease inhibitor cocktail (Nacalai Tesque). .. The supernatant (200 µl) was transferred to a 0.2-ml eight-tube strip containing GST-tagged anti–GFP Nb bound to glutathione–Sepharose 4B beads (∼5 µl bed volume of the beads) and incubated for 1 h at 4°C with constant rotation of the tube.

Protease Inhibitor:

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Cell Culture:

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Article Title: Interaction of heterotrimeric kinesin-II with IFT-B–connecting tetramer is crucial for ciliogenesis
Article Snippet: HEK293T cells on a six-well plate (∼1.6 × 106 cells) were cotransfected with expression vectors for EGFP and mChe or tRFP fusion constructs using 20 µg polyethylenimine max (Polysciences) and cultured for 24 h in DMEM with high glucose (Nacalai Tesque) supplemented with 5% FBS. .. The transfected cells were lysed in 250 µl HMDEKN cell-lysis buffer (10 mM Hepes, pH 7.4, 5 mM MgSO4 , 1 mM DTT, 0.5 mM EDTA, 25 mM KCl, and 0.5% NP-40) containing protease inhibitor cocktail (Nacalai Tesque).

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Article Snippet: .. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM (1.0 g/L glucose) with l -glutamine and sodium pyruvate; Nacalai Tesque, Kyoto, Japan) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco-BRL, Grand Island, NY, USA) in a humidified incubator with 5% CO2 at 37 °C. ..

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Article Snippet: Paragraph title: CR cell culture ... After adding the ROCK inhibitor Y-27632 at a final concentration of 10 μM, complete DMEM consists of 500 ml of DMEM, 50 ml of FBS (Cosmo Bio, Tokyo, Japan), 5.5 ml of l-glutamine (Nacalai Tesque) and 5.5 ml of penicillin-/streptomycin (Nacalai Tesque).

Article Title: Exogenous Cripto-1 Suppresses Self-Renewal of Cancer Stem Cell Model
Article Snippet: .. Cell Culture Mouse LLC cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in Dulbecco’s Modified Eagle’s Medium-high glucose (DMEM, Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA) and 100 U/mL penicillin/streptomycin (Wako, Osaka, Japan). miPS cells (iPS MEF-Ng-20D-17) (Riken Cell Bank, Ibaraki, Japan) [ ] were cultured in miPS media (DMEM containing 15% FBS, 0.1 mM MEM Non-Essential amino acids, (NEAA, Thermo Fisher Scientific, Waltham, MA, USA), 2 mM L-Glutamine (Nacalai Tesque, Kyoto, Japan), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), 50 U/mL penicillin/ streptomycin (Wako, Osaka, Japan) and 1000 U/mL of Leukemia inhibitory factor (LIF, Merck Millipore, Burlington, MA, USA)) on a feeder layer of mitomycin-treated mouse embryonic fibroblast (MEF) cells (REPROCELL Inc., Kanagawa, Japan). miPS-LLCcm cells have been established as a mouse cancer stem cell model by converted from miPS cells [ , , , , ]. ..

Article Title: Expression of Coxsackievirus and Adenovirus Receptor Separates Hematopoietic and Cardiac Progenitor Cells in Fetal Liver Kinase 1-Expressing Mesoderm
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Article Title: Induction of Expandable Tissue-Specific Progenitor Cells from Human Pancreatic Tissue through Transient Expression of Defined Factors
Article Snippet: .. Cell Culture iPS and iTP cells (induced cells described above) were maintained on an MEF feeder layer in DMEM-F12 (Sigma-Aldrich), 2 mM L-glutamine (Nacalai Tesque, Kyoto, Japan), 1:100 dilution of nonessential amino acids (Life Technologies), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), 5 ng/mL bFGF (Repro CELL), and penicillin/streptomycin (Sigma-Aldrich). .. For passaging, iPS/iTP colonies were dissociated with Dissociation Solution for human ESCs/iPSCs (Riken CDB, Kobe, Japan) and split at ratios between 1:3 and 1:6.

Mutagenesis:

Article Title: The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice *The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice * S⃞
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other:

Article Title: Inhibitory effects of rubratoxin A, a potent inhibitor of protein phosphatase 2, on the Ca2+-dependent contraction of skinned carotid artery from guinea pig
Article Snippet: Solutions and chemical NES contained 150 mM NaCl, 4 mM KCl, 2 mM CaCl2 , 1 mM MgCl2 , 10 mM glucose, 5 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulphonic acid (HEPES, Nacalai Tesque, Kyoto, Japan), 50 μU/ml insulin (Sigma), and pH was adjusted with Tris (hydroxymethyl) aminomethane (Tris; Nacalai Tesque) /H2 O to pH 7.40 at 25 °C.

Article Title: A Phosphofructokinase Homolog from Pyrobaculum calidifontis Displays Kinase Activity towards Pyrimidine Nucleosides and Ribose 1-Phosphate
Article Snippet: Tris, N , N -bis(2-hydroxyethyl)glycine (Bicine), 2-( N -morpholino)ethanesulfonic acid (MES), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), HEPES, N -[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (Tricine), 2-(cyclohexylamino)ethanesulfonic acid (CHES), lactose, 2-deoxyribose, myo -inositol, AMP, and dAMP were purchased from Nacalai Tesque.

Polymerase Chain Reaction:

Article Title: Ethanol extracts of chickpeas alter the total lipid content and expression levels of genes related to fatty acid metabolism in mouse 3T3-L1 adipocytes
Article Snippet: Dulbecco's modified Eagle's medium (DMEM) with L-glutamine, sodium pyruvate and non-fat dry milk were obtained from Nacalai Tesque (Kyoto, Japan). .. Primers and SYBR-Green for reverse transcription-quantitative PCR (RT-qPCR) and Tris-buffered saline with Tween-20 (TBS-T) tablets, pH 7.6 (100 tablets) were purchased from Takara Bio Inc. (Shiga, Japan).

Quantitative RT-PCR:

Article Title: Ethanol extracts of chickpeas alter the total lipid content and expression levels of genes related to fatty acid metabolism in mouse 3T3-L1 adipocytes
Article Snippet: Dulbecco's modified Eagle's medium (DMEM) with L-glutamine, sodium pyruvate and non-fat dry milk were obtained from Nacalai Tesque (Kyoto, Japan). .. Primers and SYBR-Green for reverse transcription-quantitative PCR (RT-qPCR) and Tris-buffered saline with Tween-20 (TBS-T) tablets, pH 7.6 (100 tablets) were purchased from Takara Bio Inc. (Shiga, Japan).

Recombinant:

Article Title: E-Cadherin Promotes Incorporation of Mouse Epiblast Stem Cells into Normal Development
Article Snippet: .. The endoderm layer of the embryos was peeled off manually with glass needles, and transferred into mEpiSC medium consisting of DMEM-F12, 20% knock-out serum replacement, 1 mM sodium pyruvate, 1x non-essential amino acids (Nacalai Tesque), 10−4 M 2-ME, 10 ng/mL activin A (Wako Pure Chemical Industries, Ltd.), and 10 ng/mL human recombinant FGF2 (Peprotech) on mitomycin C-treated mouse embryonic fibroblasts. .. To purify stem cell colonies, differentiated cells surrounding the stem cells were discarded by manually picking under a microscope (until passage 5).

MTT Assay:

Article Title: A Human ABC Transporter ABCC4 Gene SNP (rs11568658, 559 G > T, G187W) Reduces ABCC4-Dependent Drug Resistance
Article Snippet: .. The following reagents and drugs were purchased from the commercial sources indicated in parentheses: antibiotic-antimycotic cocktail solution, l -glutamine, high-glucose Dulbecco’s modified Eagle’s medium (DMEM), hygromycin B (Nacalai Tesque, Inc., Kyoto, Japan); fetal bovine serum (FBS) (Equitech-Bio, Inc., Kerrville, TX, USA); and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT reagent) (Sigma-Aldrich Co., St. Louis, MO, USA). ..

In Vivo:

Article Title: Exogenous Cripto-1 Suppresses Self-Renewal of Cancer Stem Cell Model
Article Snippet: Cell Culture Mouse LLC cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in Dulbecco’s Modified Eagle’s Medium-high glucose (DMEM, Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA) and 100 U/mL penicillin/streptomycin (Wako, Osaka, Japan). miPS cells (iPS MEF-Ng-20D-17) (Riken Cell Bank, Ibaraki, Japan) [ ] were cultured in miPS media (DMEM containing 15% FBS, 0.1 mM MEM Non-Essential amino acids, (NEAA, Thermo Fisher Scientific, Waltham, MA, USA), 2 mM L-Glutamine (Nacalai Tesque, Kyoto, Japan), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), 50 U/mL penicillin/ streptomycin (Wako, Osaka, Japan) and 1000 U/mL of Leukemia inhibitory factor (LIF, Merck Millipore, Burlington, MA, USA)) on a feeder layer of mitomycin-treated mouse embryonic fibroblast (MEF) cells (REPROCELL Inc., Kanagawa, Japan). miPS-LLCcm cells have been established as a mouse cancer stem cell model by converted from miPS cells [ , , , , ]. .. Differentiation potential was confirmed by the morphological change into the epithelial-like flat cell shape in adhesive conditions and into CD31 positive vascular endothelial cells forming tubes in Matrigel, as well as in vivo. miPS-LLCcm cells are highly tumorigenic when subcutaneously transplanted into Balb/c nude mice.

Fluorescence:

Article Title: Interaction of heterotrimeric kinesin-II with IFT-B–connecting tetramer is crucial for ciliogenesis
Article Snippet: The transfected cells were lysed in 250 µl HMDEKN cell-lysis buffer (10 mM Hepes, pH 7.4, 5 mM MgSO4 , 1 mM DTT, 0.5 mM EDTA, 25 mM KCl, and 0.5% NP-40) containing protease inhibitor cocktail (Nacalai Tesque). .. After centrifugation of the tube at 2,000 g for 30 s, the precipitated beads were washed three times with 180 µl lysis buffer, transferred to a 96-well plate, and observed using an all-in-one type microscope (BZ-8000; Keyence) with a 20× 0.75 NA objective lens under fixed conditions (sensitivity ISO 400; exposure 1/30 s for green fluorescence; and sensitivity ISO 800, exposure 1/10 s for red fluorescence), unless otherwise noted.

Article Title: Expression of Coxsackievirus and Adenovirus Receptor Separates Hematopoietic and Cardiac Progenitor Cells in Fetal Liver Kinase 1-Expressing Mesoderm
Article Snippet: To form EBs, the cells were then suspended in differentiation medium (Dulbecco’s modified Eagle’s medium; Wako Chemical, Osaka, Japan, ) supplemented with 15% FBS, NEAA, penicillin/streptomycin, 2 mM l -glutamine, and 100 μM 2-mercaptoethanol (2-ME; Nacalai Tesque Inc., Kyoto, Japan, ) and plated on a round-bottom Lipidure-coated 96-well plate (Thermo Fisher Scientific, Yokohama, Japan, ) at 3 × 103 cells (ESCs) or 1 × 103 cells (iPSCs) per well. .. To evaluate the differentiation potential of day 7 EB-derived cells, the EBs were harvested and subjected to fluorescence-activated cell sorting (FACS).

Passaging:

Article Title: Induction of Expandable Tissue-Specific Progenitor Cells from Human Pancreatic Tissue through Transient Expression of Defined Factors
Article Snippet: Cell Culture iPS and iTP cells (induced cells described above) were maintained on an MEF feeder layer in DMEM-F12 (Sigma-Aldrich), 2 mM L-glutamine (Nacalai Tesque, Kyoto, Japan), 1:100 dilution of nonessential amino acids (Life Technologies), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), 5 ng/mL bFGF (Repro CELL), and penicillin/streptomycin (Sigma-Aldrich). .. For passaging, iPS/iTP colonies were dissociated with Dissociation Solution for human ESCs/iPSCs (Riken CDB, Kobe, Japan) and split at ratios between 1:3 and 1:6.

Isolation:

Article Title: Moderate Hypoxia Induces β-Cell Dysfunction with HIF-1–Independent Gene Expression Changes
Article Snippet: Paragraph title: Isolation of pancreatic islets ... They were then cultured in RPMI1640 medium supplemented with 11 mM glucose, 10% (v/v) fetal bovine serum, 0.1% (v/v) penicillin/streptomycin, 50 µM β-mercaptoethanol, 10 mM HEPES (Nacalai Tesque, Kyoto, Japan), and 1 mM sodium pyruvate (Nacalai Tesque) at 37°C in 5% CO2 , 95% air.

Article Title: Exogenous nitric oxide stimulates the odontogenic differentiation of rat dental pulp stem cells
Article Snippet: Paragraph title: Isolation and culture of rat dental pulp stem cells (rDPSCs) ... The remaining adherent cells were incubated in alpha-modified minimum essential medium (αMEM; Thermo Fisher Scientific, Waltham, MA) supplemented with 20% fetal bovine serum (FBS; Equitech-Bio, Kerrville, TX), 100 µM L-ascorbic acid 2-phosphate (Wako Pure Chemicals, Osaka, Japan), 2 mM L-glutamine (Nacalai Tesque, Kyoto, Japan), 55 mM 2-mercaptoethanol (2-ME; Thermo Fisher Scientific), and penicillin/streptomycin (100 U/mL penicillin and 100 g/mL streptomycin; Nacalai Tesque) at 37 °C and 5% CO2 .

Growth Assay:

Article Title: The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice *The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice * S⃞
Article Snippet: These cells were cultured in RPMI 1640 medium (Nissui Seiyaku, Tokyo, Japan) supplemented with 10% fetal bovine serum (BioWest), 100 μg/ml l -glutamine (Nacalai Tesque, Tokyo, Japan), 100 μg/ml penicillin (Nacalai Tesque), 100 μg/ml streptomycin (Nacalai Tesque), and 5 units/ml Epo. .. BaF3 Cell Growth Assay —Transduced and exponentially growing BaF3 cells were washed twice with PBS and incubated with RPMI 1640 medium supplemented with 1% fetal bovine serum and 100 mg/ml l -glutamine in the presence or absence of Epo (5 units/ml) for the indicated times.

Microscopy:

Article Title: Interaction of heterotrimeric kinesin-II with IFT-B–connecting tetramer is crucial for ciliogenesis
Article Snippet: The transfected cells were lysed in 250 µl HMDEKN cell-lysis buffer (10 mM Hepes, pH 7.4, 5 mM MgSO4 , 1 mM DTT, 0.5 mM EDTA, 25 mM KCl, and 0.5% NP-40) containing protease inhibitor cocktail (Nacalai Tesque). .. After centrifugation of the tube at 2,000 g for 30 s, the precipitated beads were washed three times with 180 µl lysis buffer, transferred to a 96-well plate, and observed using an all-in-one type microscope (BZ-8000; Keyence) with a 20× 0.75 NA objective lens under fixed conditions (sensitivity ISO 400; exposure 1/30 s for green fluorescence; and sensitivity ISO 800, exposure 1/10 s for red fluorescence), unless otherwise noted.

Article Title: E-Cadherin Promotes Incorporation of Mouse Epiblast Stem Cells into Normal Development
Article Snippet: The endoderm layer of the embryos was peeled off manually with glass needles, and transferred into mEpiSC medium consisting of DMEM-F12, 20% knock-out serum replacement, 1 mM sodium pyruvate, 1x non-essential amino acids (Nacalai Tesque), 10−4 M 2-ME, 10 ng/mL activin A (Wako Pure Chemical Industries, Ltd.), and 10 ng/mL human recombinant FGF2 (Peprotech) on mitomycin C-treated mouse embryonic fibroblasts. .. To purify stem cell colonies, differentiated cells surrounding the stem cells were discarded by manually picking under a microscope (until passage 5).

Mouse Assay:

Article Title: Moderate Hypoxia Induces β-Cell Dysfunction with HIF-1–Independent Gene Expression Changes
Article Snippet: Isolation of pancreatic islets Pancreatic islets were isolated from C57BL6J mice (18–20 weeks old) by collagenase digestion as described previously . .. They were then cultured in RPMI1640 medium supplemented with 11 mM glucose, 10% (v/v) fetal bovine serum, 0.1% (v/v) penicillin/streptomycin, 50 µM β-mercaptoethanol, 10 mM HEPES (Nacalai Tesque, Kyoto, Japan), and 1 mM sodium pyruvate (Nacalai Tesque) at 37°C in 5% CO2 , 95% air.

Article Title: E-Cadherin Promotes Incorporation of Mouse Epiblast Stem Cells into Normal Development
Article Snippet: Briefly, female and male 129+ter/SvJcl mice (purchased from Clea Japan Inc.) were mated, and 6.5E embryos were collected from the uterus. .. The endoderm layer of the embryos was peeled off manually with glass needles, and transferred into mEpiSC medium consisting of DMEM-F12, 20% knock-out serum replacement, 1 mM sodium pyruvate, 1x non-essential amino acids (Nacalai Tesque), 10−4 M 2-ME, 10 ng/mL activin A (Wako Pure Chemical Industries, Ltd.), and 10 ng/mL human recombinant FGF2 (Peprotech) on mitomycin C-treated mouse embryonic fibroblasts.

Article Title: Exogenous Cripto-1 Suppresses Self-Renewal of Cancer Stem Cell Model
Article Snippet: Cell Culture Mouse LLC cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in Dulbecco’s Modified Eagle’s Medium-high glucose (DMEM, Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA) and 100 U/mL penicillin/streptomycin (Wako, Osaka, Japan). miPS cells (iPS MEF-Ng-20D-17) (Riken Cell Bank, Ibaraki, Japan) [ ] were cultured in miPS media (DMEM containing 15% FBS, 0.1 mM MEM Non-Essential amino acids, (NEAA, Thermo Fisher Scientific, Waltham, MA, USA), 2 mM L-Glutamine (Nacalai Tesque, Kyoto, Japan), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), 50 U/mL penicillin/ streptomycin (Wako, Osaka, Japan) and 1000 U/mL of Leukemia inhibitory factor (LIF, Merck Millipore, Burlington, MA, USA)) on a feeder layer of mitomycin-treated mouse embryonic fibroblast (MEF) cells (REPROCELL Inc., Kanagawa, Japan). miPS-LLCcm cells have been established as a mouse cancer stem cell model by converted from miPS cells [ , , , , ]. .. Differentiation potential was confirmed by the morphological change into the epithelial-like flat cell shape in adhesive conditions and into CD31 positive vascular endothelial cells forming tubes in Matrigel, as well as in vivo. miPS-LLCcm cells are highly tumorigenic when subcutaneously transplanted into Balb/c nude mice.

Co-Culture Assay:

Article Title: Clinical implications of drug-screening assay for recurrent metastatic hormone receptor-positive, human epidermal receptor 2-negative breast cancer using conditionally reprogrammed cells
Article Snippet: Before co-culture, detached and collected J2 cells were resuspended in 10 ml of DMEM and irradiated with X-ray (30 Gy). .. After adding the ROCK inhibitor Y-27632 at a final concentration of 10 μM, complete DMEM consists of 500 ml of DMEM, 50 ml of FBS (Cosmo Bio, Tokyo, Japan), 5.5 ml of l-glutamine (Nacalai Tesque) and 5.5 ml of penicillin-/streptomycin (Nacalai Tesque).

Lysis:

Article Title: Interaction of heterotrimeric kinesin-II with IFT-B–connecting tetramer is crucial for ciliogenesis
Article Snippet: The transfected cells were lysed in 250 µl HMDEKN cell-lysis buffer (10 mM Hepes, pH 7.4, 5 mM MgSO4 , 1 mM DTT, 0.5 mM EDTA, 25 mM KCl, and 0.5% NP-40) containing protease inhibitor cocktail (Nacalai Tesque). .. After centrifugation of the tube at 2,000 g for 30 s, the precipitated beads were washed three times with 180 µl lysis buffer, transferred to a 96-well plate, and observed using an all-in-one type microscope (BZ-8000; Keyence) with a 20× 0.75 NA objective lens under fixed conditions (sensitivity ISO 400; exposure 1/30 s for green fluorescence; and sensitivity ISO 800, exposure 1/10 s for red fluorescence), unless otherwise noted.

Purification:

Article Title: Ethanol extracts of chickpeas alter the total lipid content and expression levels of genes related to fatty acid metabolism in mouse 3T3-L1 adipocytes
Article Snippet: Dulbecco's modified Eagle's medium (DMEM) with L-glutamine, sodium pyruvate and non-fat dry milk were obtained from Nacalai Tesque (Kyoto, Japan). .. Gentamicin reagent solution (gentamicin sulfate, 10 mg/ml), trypsin-EDTA (0.25% trypsin, 1 mM EDTA • 4Na, X1), calf serum, the TRIzol plus RNA purification kit, the superscript VILO cDNA synthesis kit and Prolong Gold Antifade Reagent were purchased from Invitrogen (Carlsbad, CA, USA).

Irradiation:

Article Title: Clinical implications of drug-screening assay for recurrent metastatic hormone receptor-positive, human epidermal receptor 2-negative breast cancer using conditionally reprogrammed cells
Article Snippet: Before co-culture, detached and collected J2 cells were resuspended in 10 ml of DMEM and irradiated with X-ray (30 Gy). .. After adding the ROCK inhibitor Y-27632 at a final concentration of 10 μM, complete DMEM consists of 500 ml of DMEM, 50 ml of FBS (Cosmo Bio, Tokyo, Japan), 5.5 ml of l-glutamine (Nacalai Tesque) and 5.5 ml of penicillin-/streptomycin (Nacalai Tesque).

Cell Attachment Assay:

Article Title: Exogenous nitric oxide stimulates the odontogenic differentiation of rat dental pulp stem cells
Article Snippet: The cells were seeded in 100-mm culture dishes and were incubated for 3 hours at 37 °C to allow cell attachment. .. The remaining adherent cells were incubated in alpha-modified minimum essential medium (αMEM; Thermo Fisher Scientific, Waltham, MA) supplemented with 20% fetal bovine serum (FBS; Equitech-Bio, Kerrville, TX), 100 µM L-ascorbic acid 2-phosphate (Wako Pure Chemicals, Osaka, Japan), 2 mM L-glutamine (Nacalai Tesque, Kyoto, Japan), 55 mM 2-mercaptoethanol (2-ME; Thermo Fisher Scientific), and penicillin/streptomycin (100 U/mL penicillin and 100 g/mL streptomycin; Nacalai Tesque) at 37 °C and 5% CO2 .

In Vitro:

Article Title: Expression of Coxsackievirus and Adenovirus Receptor Separates Hematopoietic and Cardiac Progenitor Cells in Fetal Liver Kinase 1-Expressing Mesoderm
Article Snippet: Paragraph title: In Vitro Differentiation of Mouse ESCs and iPSCs ... To form EBs, the cells were then suspended in differentiation medium (Dulbecco’s modified Eagle’s medium; Wako Chemical, Osaka, Japan, ) supplemented with 15% FBS, NEAA, penicillin/streptomycin, 2 mM l -glutamine, and 100 μM 2-mercaptoethanol (2-ME; Nacalai Tesque Inc., Kyoto, Japan, ) and plated on a round-bottom Lipidure-coated 96-well plate (Thermo Fisher Scientific, Yokohama, Japan, ) at 3 × 103 cells (ESCs) or 1 × 103 cells (iPSCs) per well.

CCK-8 Assay:

Article Title: In vitro and in vivo characterization of anti-malarial acylphenoxazine derivatives prepared from basic blue 3
Article Snippet: The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM (1.0 g/L glucose) with l -glutamine and sodium pyruvate; Nacalai Tesque, Kyoto, Japan) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco-BRL, Grand Island, NY, USA) in a humidified incubator with 5% CO2 at 37 °C. .. Basic blue 3, ITT-001 to 006, and CQ at gradually decreasing concentrations (50, 15, 5, 1.5, 0.5, 0.15, 0.05, and 0.015 μM) were added to the cell culture after 24 h and the cells were subsequently cultured for 48 h. Cell viability was measured using a Cell Counting Kit-8 (Dojindo) according to the manufacturer’s instructions.

Knock-Out:

Article Title: E-Cadherin Promotes Incorporation of Mouse Epiblast Stem Cells into Normal Development
Article Snippet: .. The endoderm layer of the embryos was peeled off manually with glass needles, and transferred into mEpiSC medium consisting of DMEM-F12, 20% knock-out serum replacement, 1 mM sodium pyruvate, 1x non-essential amino acids (Nacalai Tesque), 10−4 M 2-ME, 10 ng/mL activin A (Wako Pure Chemical Industries, Ltd.), and 10 ng/mL human recombinant FGF2 (Peprotech) on mitomycin C-treated mouse embryonic fibroblasts. .. To purify stem cell colonies, differentiated cells surrounding the stem cells were discarded by manually picking under a microscope (until passage 5).

Concentration Assay:

Article Title: Clinical implications of drug-screening assay for recurrent metastatic hormone receptor-positive, human epidermal receptor 2-negative breast cancer using conditionally reprogrammed cells
Article Snippet: .. After adding the ROCK inhibitor Y-27632 at a final concentration of 10 μM, complete DMEM consists of 500 ml of DMEM, 50 ml of FBS (Cosmo Bio, Tokyo, Japan), 5.5 ml of l-glutamine (Nacalai Tesque) and 5.5 ml of penicillin-/streptomycin (Nacalai Tesque). ..

Article Title: E-Cadherin Promotes Incorporation of Mouse Epiblast Stem Cells into Normal Development
Article Snippet: The endoderm layer of the embryos was peeled off manually with glass needles, and transferred into mEpiSC medium consisting of DMEM-F12, 20% knock-out serum replacement, 1 mM sodium pyruvate, 1x non-essential amino acids (Nacalai Tesque), 10−4 M 2-ME, 10 ng/mL activin A (Wako Pure Chemical Industries, Ltd.), and 10 ng/mL human recombinant FGF2 (Peprotech) on mitomycin C-treated mouse embryonic fibroblasts. .. Cells were routinely passaged on fibronectin (BD Biosciences)-coated dishes every 3 days using accutase (Sigma) or 1 mg/mL collagenase type-IV (Gibco) dissolved in DMEM-F12 at a concentration of 10 mg/mL as a stock solution.

Cell Counting:

Article Title: The dominant role of proofreading exonuclease activity of replicative polymerase ε in cellular tolerance to cytarabine (Ara-C)
Article Snippet: DT40 and TK6 cell culture Culture conditions for DT40 cells, cell counting and cell cycle analysis have been described previously [ ]. .. TK6 cells were cultured in an RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% heat-inactivated horse serum (HS) (GIBCO, lot No. 2017-06), 0.1mM Sodium pyruvate, L-glutamine (Nacalai Tesque), 50 U/mL penicillin, and 50 μg/mL streptomycin (Nacalai Tesque).

Marker:

Article Title: Exogenous Cripto-1 Suppresses Self-Renewal of Cancer Stem Cell Model
Article Snippet: Cell Culture Mouse LLC cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in Dulbecco’s Modified Eagle’s Medium-high glucose (DMEM, Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA) and 100 U/mL penicillin/streptomycin (Wako, Osaka, Japan). miPS cells (iPS MEF-Ng-20D-17) (Riken Cell Bank, Ibaraki, Japan) [ ] were cultured in miPS media (DMEM containing 15% FBS, 0.1 mM MEM Non-Essential amino acids, (NEAA, Thermo Fisher Scientific, Waltham, MA, USA), 2 mM L-Glutamine (Nacalai Tesque, Kyoto, Japan), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), 50 U/mL penicillin/ streptomycin (Wako, Osaka, Japan) and 1000 U/mL of Leukemia inhibitory factor (LIF, Merck Millipore, Burlington, MA, USA)) on a feeder layer of mitomycin-treated mouse embryonic fibroblast (MEF) cells (REPROCELL Inc., Kanagawa, Japan). miPS-LLCcm cells have been established as a mouse cancer stem cell model by converted from miPS cells [ , , , , ]. .. In addition, miPS-LLCcm cells express the marker genes, such as Nanog, Rex1, Eras, Esg1, Cripto-1, CD44 and ALDH1, which are considered associated with cancer stem cell properties. miPS-LLCcm cells were cultured in miPS media without LIF.

FACS:

Article Title: Expression of Coxsackievirus and Adenovirus Receptor Separates Hematopoietic and Cardiac Progenitor Cells in Fetal Liver Kinase 1-Expressing Mesoderm
Article Snippet: To form EBs, the cells were then suspended in differentiation medium (Dulbecco’s modified Eagle’s medium; Wako Chemical, Osaka, Japan, ) supplemented with 15% FBS, NEAA, penicillin/streptomycin, 2 mM l -glutamine, and 100 μM 2-mercaptoethanol (2-ME; Nacalai Tesque Inc., Kyoto, Japan, ) and plated on a round-bottom Lipidure-coated 96-well plate (Thermo Fisher Scientific, Yokohama, Japan, ) at 3 × 103 cells (ESCs) or 1 × 103 cells (iPSCs) per well. .. To evaluate the differentiation potential of day 7 EB-derived cells, the EBs were harvested and subjected to fluorescence-activated cell sorting (FACS).

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  • 99
    Nacalai hepes
    Effects of pH and temperature on the kinase activity of Pcal_0041. (A) Effect of pH. Kinase activity of Pcal_0041 was examined at 85°C in various buffers: ■, <t>MES-NaOH</t> (pH 5.5 to 7.0); □, <t>HEPES-NaOH</t> (pH 7.0 to 8.0); ●, Tricine-NaOH (pH 8.0 to 9.0); ○, CHES-NaOH (9.0 to 10); ▲, CAPS-NaOH (10.0 to 11.0). (B) Effect of temperature. Enzyme activity was examined in HEPES-NaOH buffer (pH 8.0) at various temperatures from 50 to 90°C. (C) Thermostability of Pcal_0041 at 100°C. Pcal_0041 was heated at 100°C for various intervals of time and the residual activity was examined at 85°C and pH 8.0. All buffers were prepared at the temperatures of use. Each point is the average from at least three measurements. SD values are indicated with bars. (D) Circular dichroism studies on recombinant Pcal_0041 protein. CD spectra from 190 to 280 nm were measured at various temperatures: ◆, 30°C; ■, 50°C; ▲, 70°C; ✖, 80°C; ○, 90°C.
    Hepes, supplied by Nacalai, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Nacalai hmdekn cell lysis buffer
    Effects of pH and temperature on the kinase activity of Pcal_0041. (A) Effect of pH. Kinase activity of Pcal_0041 was examined at 85°C in various buffers: ■, <t>MES-NaOH</t> (pH 5.5 to 7.0); □, <t>HEPES-NaOH</t> (pH 7.0 to 8.0); ●, Tricine-NaOH (pH 8.0 to 9.0); ○, CHES-NaOH (9.0 to 10); ▲, CAPS-NaOH (10.0 to 11.0). (B) Effect of temperature. Enzyme activity was examined in HEPES-NaOH buffer (pH 8.0) at various temperatures from 50 to 90°C. (C) Thermostability of Pcal_0041 at 100°C. Pcal_0041 was heated at 100°C for various intervals of time and the residual activity was examined at 85°C and pH 8.0. All buffers were prepared at the temperatures of use. Each point is the average from at least three measurements. SD values are indicated with bars. (D) Circular dichroism studies on recombinant Pcal_0041 protein. CD spectra from 190 to 280 nm were measured at various temperatures: ◆, 30°C; ■, 50°C; ▲, 70°C; ✖, 80°C; ○, 90°C.
    Hmdekn Cell Lysis Buffer, supplied by Nacalai, used in various techniques. Bioz Stars score: 98/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Nacalai 2 4 2 hydroxyethyl 1 piperazinyl ethanesulphonic acid
    Effects of pH and temperature on the kinase activity of Pcal_0041. (A) Effect of pH. Kinase activity of Pcal_0041 was examined at 85°C in various buffers: ■, <t>MES-NaOH</t> (pH 5.5 to 7.0); □, <t>HEPES-NaOH</t> (pH 7.0 to 8.0); ●, Tricine-NaOH (pH 8.0 to 9.0); ○, CHES-NaOH (9.0 to 10); ▲, CAPS-NaOH (10.0 to 11.0). (B) Effect of temperature. Enzyme activity was examined in HEPES-NaOH buffer (pH 8.0) at various temperatures from 50 to 90°C. (C) Thermostability of Pcal_0041 at 100°C. Pcal_0041 was heated at 100°C for various intervals of time and the residual activity was examined at 85°C and pH 8.0. All buffers were prepared at the temperatures of use. Each point is the average from at least three measurements. SD values are indicated with bars. (D) Circular dichroism studies on recombinant Pcal_0041 protein. CD spectra from 190 to 280 nm were measured at various temperatures: ◆, 30°C; ■, 50°C; ▲, 70°C; ✖, 80°C; ○, 90°C.
    2 4 2 Hydroxyethyl 1 Piperazinyl Ethanesulphonic Acid, supplied by Nacalai, used in various techniques. Bioz Stars score: 83/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effects of pH and temperature on the kinase activity of Pcal_0041. (A) Effect of pH. Kinase activity of Pcal_0041 was examined at 85°C in various buffers: ■, MES-NaOH (pH 5.5 to 7.0); □, HEPES-NaOH (pH 7.0 to 8.0); ●, Tricine-NaOH (pH 8.0 to 9.0); ○, CHES-NaOH (9.0 to 10); ▲, CAPS-NaOH (10.0 to 11.0). (B) Effect of temperature. Enzyme activity was examined in HEPES-NaOH buffer (pH 8.0) at various temperatures from 50 to 90°C. (C) Thermostability of Pcal_0041 at 100°C. Pcal_0041 was heated at 100°C for various intervals of time and the residual activity was examined at 85°C and pH 8.0. All buffers were prepared at the temperatures of use. Each point is the average from at least three measurements. SD values are indicated with bars. (D) Circular dichroism studies on recombinant Pcal_0041 protein. CD spectra from 190 to 280 nm were measured at various temperatures: ◆, 30°C; ■, 50°C; ▲, 70°C; ✖, 80°C; ○, 90°C.

    Journal: Journal of Bacteriology

    Article Title: A Phosphofructokinase Homolog from Pyrobaculum calidifontis Displays Kinase Activity towards Pyrimidine Nucleosides and Ribose 1-Phosphate

    doi: 10.1128/JB.00284-18

    Figure Lengend Snippet: Effects of pH and temperature on the kinase activity of Pcal_0041. (A) Effect of pH. Kinase activity of Pcal_0041 was examined at 85°C in various buffers: ■, MES-NaOH (pH 5.5 to 7.0); □, HEPES-NaOH (pH 7.0 to 8.0); ●, Tricine-NaOH (pH 8.0 to 9.0); ○, CHES-NaOH (9.0 to 10); ▲, CAPS-NaOH (10.0 to 11.0). (B) Effect of temperature. Enzyme activity was examined in HEPES-NaOH buffer (pH 8.0) at various temperatures from 50 to 90°C. (C) Thermostability of Pcal_0041 at 100°C. Pcal_0041 was heated at 100°C for various intervals of time and the residual activity was examined at 85°C and pH 8.0. All buffers were prepared at the temperatures of use. Each point is the average from at least three measurements. SD values are indicated with bars. (D) Circular dichroism studies on recombinant Pcal_0041 protein. CD spectra from 190 to 280 nm were measured at various temperatures: ◆, 30°C; ■, 50°C; ▲, 70°C; ✖, 80°C; ○, 90°C.

    Article Snippet: Tris, N , N -bis(2-hydroxyethyl)glycine (Bicine), 2-( N -morpholino)ethanesulfonic acid (MES), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), HEPES, N -[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (Tricine), 2-(cyclohexylamino)ethanesulfonic acid (CHES), lactose, 2-deoxyribose, myo -inositol, AMP, and dAMP were purchased from Nacalai Tesque.

    Techniques: Activity Assay, Recombinant