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Thermo Fisher n2
N2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Cell Isolation:

Article Title: Differentiation of Multipotent Vascular Stem Cells Contributes to Vascular Diseases
Article Snippet: Paragraph title: Cell isolation ... The cells were cultured in DMEM with 10% FBS (Thermo Fisher Scientific Inc.), or in DMEM with 2% CEE (MP Biomedical, Inc.), 1% FBS, 1% N2 (Invitrogen Corp.), 2% B27 (Invitrogen Corp.), 100 nM retinoic acid (RA) (Sigma-Aldrich, Inc.), 50 nM 2-mercaptoethanol (2ME) (Sigma-Aldrich, Inc.), 1% P/S and 20 ng/ml bFGF (R & D Systems, Inc.) (Maintenance medium).

Cytometry:

Article Title: Analysis of transcriptional variability in a large human iPSC library reveals genetic and non-genetic determinants of heterogeneity
Article Snippet: The cells were then cultured for 3 days in DMEM/F12 supplemented with B27 and N2 (Thermo), 5μM CHIR-99021 (Selleckchem) and 25 ng/ml BMP-4 (Peprotech). .. Endothelial differentiation was performed in triplicates and calculated as CD31+/CD144+ cell percentage through flow cytometry.

Real-time Polymerase Chain Reaction:

Article Title: Focal Adhesion Kinase Inhibition Contributes to Tumor Cell Survival and Motility in Neuroblastoma Patient-Derived Xenografts
Article Snippet: Individual cells were obtained by dissociating the COA3 and COA6 xenograft tumors using a Tumor Dissociation Kit (Miltenyi Biotec, San Diego, CA) per manufacturer’s protocol, and resuspended in neurobasal medium (NB) (Life Technologies, Carlsbad, CA) supplemented with B-27 without Vitamin A (Life Technologies), N2 (Life Technologies), amphotericin B (250 μg/mL), gentamicin (50μg/mL), L-glutamine (2 mM), epidermal growth factor (10 ng/mL; Miltenyi Biotec), and fibroblast growth factor (10 ng/mL; Miltenyi Biotec). .. Real-time qPCR was performed to assess the percentage of human and mouse DNA contained in the COA3 and COA6 PDXs to ensure that the tumors did not harbor murine contamination (TRENDD RNA/DNA Isolation and TaqMan QPCR/Genotyping Core Facility, UAB, Birmingham, AL).

Incubation:

Article Title: Differentiation of Multipotent Vascular Stem Cells Contributes to Vascular Diseases
Article Snippet: The cells were cultured in DMEM with 10% FBS (Thermo Fisher Scientific Inc.), or in DMEM with 2% CEE (MP Biomedical, Inc.), 1% FBS, 1% N2 (Invitrogen Corp.), 2% B27 (Invitrogen Corp.), 100 nM retinoic acid (RA) (Sigma-Aldrich, Inc.), 50 nM 2-mercaptoethanol (2ME) (Sigma-Aldrich, Inc.), 1% P/S and 20 ng/ml bFGF (R & D Systems, Inc.) (Maintenance medium). .. For enzymatic digestion methods, tissues were incubated with 3 mg/ml type II collagenase (Sigma-Aldrich Inc.) in DMEM with a 1/5 (w/v) ratio of tissue (g) to enzyme solution (ml).

Infection:

Article Title: Groucho related gene 5 (GRG5) is involved in embryonic and neural stem cell state decisions
Article Snippet: Accordingly, for GRG5 overexpression, cells were infected with lentiviruses GRG5-TetO-FUW and rtTA and selected with blasticidin 2 μg/ml. .. For the differentiation assay neurospheres were dissociated and plated on Matrigel coated surface in differentiation medium containing basal medium supplemented with N2 (GIBCO) and 1% FBS for five days.

Expressing:

Article Title: Dietary fiber sources and non-starch polysaccharide-degrading enzymes modify mucin expression and the immune profile of the swine ileum
Article Snippet: .. Crypt isolation and enteroid culture Enteroids were cultured from mouse isolated crypts obtained following the protocols described by Mahe et al. [ ], with culture media formulated with advanced DMEM/F12 (Gibco, Thermo Fisher Scientific, Waltham, MA) supplemented with 2mM GlutaMax (Gibco), 10 mM HEPES (Gibco), 100 U/mL penicillin/100 μg/mL streptomycin (Gibco), 1× N2 (Gibco) and 1× B27 (Gibco) supplements, 100 ng/mL Noggin (Gibco), 50 ng/mL epithelial growth factor (R & D Systems, Minneapolis, MN), 5% R-spondin, 5% Wnt-3a conditioned media obtained from transformed Hek293 cells expressing R-spondin (a kind gift from Dr. Jason Spence, University of Michigan), and Wnt-3a L-cells (ATTCC CRL-2647). ..

Article Title: Groucho related gene 5 (GRG5) is involved in embryonic and neural stem cell state decisions
Article Snippet: To induce exogenous GRG5 expression 2 μg/ml doxycycline was used. .. For the differentiation assay neurospheres were dissociated and plated on Matrigel coated surface in differentiation medium containing basal medium supplemented with N2 (GIBCO) and 1% FBS for five days.

Modification:

Article Title: Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture
Article Snippet: Growth factor-free chemically defined medium (gfCDM) was prepared as follows: Iscove’s modified Dulbecco’s medium (IMDM; invitrogen)/Ham’s F12 medium (invitrogen) 1∶1, 1× Chemically-defined lipid concentrate (invitrogen), 450 µM monothioglycerol (Sigma), 5 mg/ml purified bovine serum albumin ( > 99% purified by crystallization; Sigma) and 15 µg/ml apo-transferrin (Sigma). .. On day 5, cell aggregates were transferred to a 10-cm bacterial-grade dish with DMEM/F12 supplemented with N2 (Invitrogen).

Article Title: Role of the JNK Pathway in Varicella-Zoster Virus Lytic Infection and Reactivation
Article Snippet: The HES (WA09/H9; WiCell)-based SOX10::GFP reporter line was cultured with mouse embryonic fibroblasts (ASF-113; Applied Stem Cell) in proliferation medium consisting of Dulbecco's modified Eagle's medium (DMEM)–F-12 (catalog no. 11330-057; Gibco) base medium supplemented with knockout serum (catalog no. 10828-028; Gibco), 2 mM l -glutamine (catalog no. 25030-081; Gibco), MEM nonessential amino acids (catalog no. 11140-050; Gibco), 5.5 mM 2-mercaptoethanol (catalog no. 21985-023; Gibco). .. Briefly, 500 nM LDN193189 (Abcam) and 10 μM SB431542 (Cayman Chemical) were added for 3 days, followed by additional treatment with Chir 99021 (Tocris Bioscience) for 7 days, with gradual medium change from HES medium to neurobasal medium containing N2 (catalog no. 17502-048; Gibco), B27 (catalog no. 12587-010; Gibco), and 2 mM l -glutamine.

Transformation Assay:

Article Title: Dietary fiber sources and non-starch polysaccharide-degrading enzymes modify mucin expression and the immune profile of the swine ileum
Article Snippet: .. Crypt isolation and enteroid culture Enteroids were cultured from mouse isolated crypts obtained following the protocols described by Mahe et al. [ ], with culture media formulated with advanced DMEM/F12 (Gibco, Thermo Fisher Scientific, Waltham, MA) supplemented with 2mM GlutaMax (Gibco), 10 mM HEPES (Gibco), 100 U/mL penicillin/100 μg/mL streptomycin (Gibco), 1× N2 (Gibco) and 1× B27 (Gibco) supplements, 100 ng/mL Noggin (Gibco), 50 ng/mL epithelial growth factor (R & D Systems, Minneapolis, MN), 5% R-spondin, 5% Wnt-3a conditioned media obtained from transformed Hek293 cells expressing R-spondin (a kind gift from Dr. Jason Spence, University of Michigan), and Wnt-3a L-cells (ATTCC CRL-2647). ..

Over Expression:

Article Title: Groucho related gene 5 (GRG5) is involved in embryonic and neural stem cell state decisions
Article Snippet: Accordingly, for GRG5 overexpression, cells were infected with lentiviruses GRG5-TetO-FUW and rtTA and selected with blasticidin 2 μg/ml. .. For the differentiation assay neurospheres were dissociated and plated on Matrigel coated surface in differentiation medium containing basal medium supplemented with N2 (GIBCO) and 1% FBS for five days.

Crystallization Assay:

Article Title: Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture
Article Snippet: Growth factor-free chemically defined medium (gfCDM) was prepared as follows: Iscove’s modified Dulbecco’s medium (IMDM; invitrogen)/Ham’s F12 medium (invitrogen) 1∶1, 1× Chemically-defined lipid concentrate (invitrogen), 450 µM monothioglycerol (Sigma), 5 mg/ml purified bovine serum albumin ( > 99% purified by crystallization; Sigma) and 15 µg/ml apo-transferrin (Sigma). .. On day 5, cell aggregates were transferred to a 10-cm bacterial-grade dish with DMEM/F12 supplemented with N2 (Invitrogen).

Transfection:

Article Title: miR-367 promotes proliferation and stem-like traits in medulloblastoma cells
Article Snippet: .. Neurosphere formation assay Cells were transfected with miR-367 or non-specific control and plated 24 h later into 96-well low-attachment plates (Corning, New York, NY), at a density of 1 × 103 cells/mL in DMEM/F12 medium containing 20 ng/mL EGF, 20 ng/mL bFGF, 1× B-27 and 1× N2 (Invitrogen). ..

Transferring:

Article Title: NAD+ supplementation rejuvenates aged gut adult stem cells, et al. NAD+ supplementation rejuvenates aged gut adult stem cells
Article Snippet: Next, we pipetted pieces of the intestine up and down in cold PBS with a pipette until most of crypts are released. .. A total of 300 isolated crypts were plated per well of a 48‐well plate and cultured in a crypt culture medium, DMEM/F12 (Thermo Scientific) supplemented by 1× N2 (Thermo Scientific), 1× B27 (Thermo Scientific), 1 mM N‐Acetyl‐L‐cysteine (Sigma), 50 ng/ml EGF (Thermo Scientific), 100 ng/ml Noggin (Peprotech), and 500 ng/ml R‐spondin (R & D or ACRO Biosystems).

Cell Culture:

Article Title: NAD+ supplementation rejuvenates aged gut adult stem cells, et al. NAD+ supplementation rejuvenates aged gut adult stem cells
Article Snippet: .. A total of 300 isolated crypts were plated per well of a 48‐well plate and cultured in a crypt culture medium, DMEM/F12 (Thermo Scientific) supplemented by 1× N2 (Thermo Scientific), 1× B27 (Thermo Scientific), 1 mM N‐Acetyl‐L‐cysteine (Sigma), 50 ng/ml EGF (Thermo Scientific), 100 ng/ml Noggin (Peprotech), and 500 ng/ml R‐spondin (R & D or ACRO Biosystems). .. When indicated, NR (ChromaDex), rapamycin (Santa cruz) or EX527 (Santa cruz) was added to crypt culture medium.

Article Title: Dietary fiber sources and non-starch polysaccharide-degrading enzymes modify mucin expression and the immune profile of the swine ileum
Article Snippet: .. Crypt isolation and enteroid culture Enteroids were cultured from mouse isolated crypts obtained following the protocols described by Mahe et al. [ ], with culture media formulated with advanced DMEM/F12 (Gibco, Thermo Fisher Scientific, Waltham, MA) supplemented with 2mM GlutaMax (Gibco), 10 mM HEPES (Gibco), 100 U/mL penicillin/100 μg/mL streptomycin (Gibco), 1× N2 (Gibco) and 1× B27 (Gibco) supplements, 100 ng/mL Noggin (Gibco), 50 ng/mL epithelial growth factor (R & D Systems, Minneapolis, MN), 5% R-spondin, 5% Wnt-3a conditioned media obtained from transformed Hek293 cells expressing R-spondin (a kind gift from Dr. Jason Spence, University of Michigan), and Wnt-3a L-cells (ATTCC CRL-2647). ..

Article Title: Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture
Article Snippet: Paragraph title: mES Cell Culture and Treatment with Soluble Factors ... On day 5, cell aggregates were transferred to a 10-cm bacterial-grade dish with DMEM/F12 supplemented with N2 (Invitrogen).

Article Title: Analysis of transcriptional variability in a large human iPSC library reveals genetic and non-genetic determinants of heterogeneity
Article Snippet: .. The cells were then cultured for 3 days in DMEM/F12 supplemented with B27 and N2 (Thermo), 5μM CHIR-99021 (Selleckchem) and 25 ng/ml BMP-4 (Peprotech). .. For the final 2 days, the cells were grown in Stempro34 (Thermo) supplemented with 200ng/ml VEGF (Peprotech) and 5μM Forskolin (LC labs).

Article Title: Differentiation of nonhuman primate embryonic stem cells along neural lineages
Article Snippet: The EBs were cultured in ESC culture medium for 48 h to permit attachment of the EBs. .. Then the culture medium was replaced with neural induction medium composed of DMEM/F12 (1:1) supplemented with N2 (Invitrogen), 100 mM MEM nonessential amino acids and basic fibroblast growth factor (bFGF) (10 ng/ml, Invitrogen).

Article Title: A culture system to study oligodendrocyte myelination-processes using engineered nanofibers
Article Snippet: .. Oligodendroglial-nanofiber cultures 250,000 rat primary oligodendroglial cells isolated from postnatal brains as described above, were cultured on substrate-coated nanofibers in chemically-defined medium composed of DMEM (Invitrogen) supplemented with B27 (Invitrogen), N2 (Invitrogen), penicillin-streptomycin (Invitrogen), N-acetyl-cysteine (Sigma-Aldrich), forskolin (Sigma-Aldrich) and 12.5 ng/ml PDGF-AA (Peprotech). .. Quantification of PDGFRα+ or MBP+ segments on fibers The distribution of nanofiber diameters (0.2–4.0 μm) on each coverslip was determined by scanning electron microscopy.

Article Title: Establishment and Identification of a CiPSC Lineage Reprogrammed from FSP-tdTomato Mouse Embryonic Fibroblasts (MEFs)
Article Snippet: Paragraph title: 2.2. Cell Culture ... The second-stage culture medium for CiPSCs (day 22–day 40) contained high glucose, DMEM (Hyclone), 1× N2 (Gibco), 2× B27 (Gibco), 1× NEAA (Gibco), 1× Gluta Max (Gibco), 1× Sodium pyruvate (Gibco), 0.1 mM β ME (Gibco), 3 μ M CHIR99021 (MCE), 1 μ M PD0325901 (MCE), and 1000 units/ml LIF (Millipore).

Article Title: Focal Adhesion Kinase Inhibition Contributes to Tumor Cell Survival and Motility in Neuroblastoma Patient-Derived Xenografts
Article Snippet: Paragraph title: Cell culture ... Individual cells were obtained by dissociating the COA3 and COA6 xenograft tumors using a Tumor Dissociation Kit (Miltenyi Biotec, San Diego, CA) per manufacturer’s protocol, and resuspended in neurobasal medium (NB) (Life Technologies, Carlsbad, CA) supplemented with B-27 without Vitamin A (Life Technologies), N2 (Life Technologies), amphotericin B (250 μg/mL), gentamicin (50μg/mL), L-glutamine (2 mM), epidermal growth factor (10 ng/mL; Miltenyi Biotec), and fibroblast growth factor (10 ng/mL; Miltenyi Biotec).

Article Title: Role of the JNK Pathway in Varicella-Zoster Virus Lytic Infection and Reactivation
Article Snippet: The HES (WA09/H9; WiCell)-based SOX10::GFP reporter line was cultured with mouse embryonic fibroblasts (ASF-113; Applied Stem Cell) in proliferation medium consisting of Dulbecco's modified Eagle's medium (DMEM)–F-12 (catalog no. 11330-057; Gibco) base medium supplemented with knockout serum (catalog no. 10828-028; Gibco), 2 mM l -glutamine (catalog no. 25030-081; Gibco), MEM nonessential amino acids (catalog no. 11140-050; Gibco), 5.5 mM 2-mercaptoethanol (catalog no. 21985-023; Gibco). .. Briefly, 500 nM LDN193189 (Abcam) and 10 μM SB431542 (Cayman Chemical) were added for 3 days, followed by additional treatment with Chir 99021 (Tocris Bioscience) for 7 days, with gradual medium change from HES medium to neurobasal medium containing N2 (catalog no. 17502-048; Gibco), B27 (catalog no. 12587-010; Gibco), and 2 mM l -glutamine.

Article Title: Differentiation of Multipotent Vascular Stem Cells Contributes to Vascular Diseases
Article Snippet: .. The cells were cultured in DMEM with 10% FBS (Thermo Fisher Scientific Inc.), or in DMEM with 2% CEE (MP Biomedical, Inc.), 1% FBS, 1% N2 (Invitrogen Corp.), 2% B27 (Invitrogen Corp.), 100 nM retinoic acid (RA) (Sigma-Aldrich, Inc.), 50 nM 2-mercaptoethanol (2ME) (Sigma-Aldrich, Inc.), 1% P/S and 20 ng/ml bFGF (R & D Systems, Inc.) (Maintenance medium). .. For enzymatic digestion methods, tissues were incubated with 3 mg/ml type II collagenase (Sigma-Aldrich Inc.) in DMEM with a 1/5 (w/v) ratio of tissue (g) to enzyme solution (ml).

Inhibition:

Article Title: Role of the JNK Pathway in Varicella-Zoster Virus Lytic Infection and Reactivation
Article Snippet: To differentiate SOX10-expressing neural crest stem cells, 6 × 104 HES cells/cm2 on Geltrex (catalog no. A1413302; Gibco) were treated with dual SMAD inhibition and canonical WNT activation, as previously described ( ). .. Briefly, 500 nM LDN193189 (Abcam) and 10 μM SB431542 (Cayman Chemical) were added for 3 days, followed by additional treatment with Chir 99021 (Tocris Bioscience) for 7 days, with gradual medium change from HES medium to neurobasal medium containing N2 (catalog no. 17502-048; Gibco), B27 (catalog no. 12587-010; Gibco), and 2 mM l -glutamine.

Recombinant:

Article Title: Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture
Article Snippet: On day 5, cell aggregates were transferred to a 10-cm bacterial-grade dish with DMEM/F12 supplemented with N2 (Invitrogen). .. ECM proteins were laminin/entactin complex (200 µg/ml, BD Biosciences), Matrigel (200 µg/ml, BD Biosciences), ultrapure laminin (100 µg/ml, BD Biosciences), fibronectin (50–100 µg/ml, BD Biosciences), human recombinant laminin (200 µg/ml, BioLamina, buffer exchanged into PBS and concentrated to 1 mg/ml using microcon (Millipore)).

Article Title: CFTR Expression Analysis for Subtyping of Human Pancreatic Cancer Organoids
Article Snippet: .. After several washing steps with DMEM/F12+++ medium, the remaining cell pellet was resuspended in GFR Matrigel (Corning) and cultivated in human PDAC organoid medium DMEM/F12+++ supplemented with Wnt3a-conditioned medium (50% v /v ), noggin-conditioned medium (10% v /v ), RSPO1-conditioned medium (10% v /v ), B27 (1x, Invitrogen), nicotinamide (10 mM, Sigma-Aldrich), gastrin (1 nM, Sigma-Aldrich), N-acetyl-L-cysteine (1 mM, Sigma-Aldrich), primocin (1 mg/ml, InvivoGen), recombinant murine epidermal growth factor (mEGF, 50 ng/ml, Invitrogen), recombinant human fibroblast growth factor 10 (hFGF10, 100 ng/ml, PeproTech), A-83-01 (0.5 μ M, Tocris Bioscience), and N2 (1x, Invitrogen). .. Immunohistochemistry (IHC) Stainings and Imaging Sections from paraffin-embedded primary PDAC tissue samples were provided by the Tumor- and Normal Tissue Bank of the Institute of Pathology, University Hospital Carl Gustav Carus.

Fluorescence:

Article Title: Role of the JNK Pathway in Varicella-Zoster Virus Lytic Infection and Reactivation
Article Snippet: Briefly, 500 nM LDN193189 (Abcam) and 10 μM SB431542 (Cayman Chemical) were added for 3 days, followed by additional treatment with Chir 99021 (Tocris Bioscience) for 7 days, with gradual medium change from HES medium to neurobasal medium containing N2 (catalog no. 17502-048; Gibco), B27 (catalog no. 12587-010; Gibco), and 2 mM l -glutamine. .. GFP-expressing cells could begin to be identified by day 7, and fluorescence-activated cell sorter (FACS) purification was performed at day 10.

Isolation:

Article Title: NAD+ supplementation rejuvenates aged gut adult stem cells, et al. NAD+ supplementation rejuvenates aged gut adult stem cells
Article Snippet: .. A total of 300 isolated crypts were plated per well of a 48‐well plate and cultured in a crypt culture medium, DMEM/F12 (Thermo Scientific) supplemented by 1× N2 (Thermo Scientific), 1× B27 (Thermo Scientific), 1 mM N‐Acetyl‐L‐cysteine (Sigma), 50 ng/ml EGF (Thermo Scientific), 100 ng/ml Noggin (Peprotech), and 500 ng/ml R‐spondin (R & D or ACRO Biosystems). .. When indicated, NR (ChromaDex), rapamycin (Santa cruz) or EX527 (Santa cruz) was added to crypt culture medium.

Article Title: Dietary fiber sources and non-starch polysaccharide-degrading enzymes modify mucin expression and the immune profile of the swine ileum
Article Snippet: .. Crypt isolation and enteroid culture Enteroids were cultured from mouse isolated crypts obtained following the protocols described by Mahe et al. [ ], with culture media formulated with advanced DMEM/F12 (Gibco, Thermo Fisher Scientific, Waltham, MA) supplemented with 2mM GlutaMax (Gibco), 10 mM HEPES (Gibco), 100 U/mL penicillin/100 μg/mL streptomycin (Gibco), 1× N2 (Gibco) and 1× B27 (Gibco) supplements, 100 ng/mL Noggin (Gibco), 50 ng/mL epithelial growth factor (R & D Systems, Minneapolis, MN), 5% R-spondin, 5% Wnt-3a conditioned media obtained from transformed Hek293 cells expressing R-spondin (a kind gift from Dr. Jason Spence, University of Michigan), and Wnt-3a L-cells (ATTCC CRL-2647). ..

Article Title: A culture system to study oligodendrocyte myelination-processes using engineered nanofibers
Article Snippet: .. Oligodendroglial-nanofiber cultures 250,000 rat primary oligodendroglial cells isolated from postnatal brains as described above, were cultured on substrate-coated nanofibers in chemically-defined medium composed of DMEM (Invitrogen) supplemented with B27 (Invitrogen), N2 (Invitrogen), penicillin-streptomycin (Invitrogen), N-acetyl-cysteine (Sigma-Aldrich), forskolin (Sigma-Aldrich) and 12.5 ng/ml PDGF-AA (Peprotech). .. Quantification of PDGFRα+ or MBP+ segments on fibers The distribution of nanofiber diameters (0.2–4.0 μm) on each coverslip was determined by scanning electron microscopy.

Article Title: Focal Adhesion Kinase Inhibition Contributes to Tumor Cell Survival and Motility in Neuroblastoma Patient-Derived Xenografts
Article Snippet: Individual cells were obtained by dissociating the COA3 and COA6 xenograft tumors using a Tumor Dissociation Kit (Miltenyi Biotec, San Diego, CA) per manufacturer’s protocol, and resuspended in neurobasal medium (NB) (Life Technologies, Carlsbad, CA) supplemented with B-27 without Vitamin A (Life Technologies), N2 (Life Technologies), amphotericin B (250 μg/mL), gentamicin (50μg/mL), L-glutamine (2 mM), epidermal growth factor (10 ng/mL; Miltenyi Biotec), and fibroblast growth factor (10 ng/mL; Miltenyi Biotec). .. Real-time qPCR was performed to assess the percentage of human and mouse DNA contained in the COA3 and COA6 PDXs to ensure that the tumors did not harbor murine contamination (TRENDD RNA/DNA Isolation and TaqMan QPCR/Genotyping Core Facility, UAB, Birmingham, AL).

Article Title: Groucho related gene 5 (GRG5) is involved in embryonic and neural stem cell state decisions
Article Snippet: Paragraph title: Embryonic NSC isolation, culture and evaluation ... For the differentiation assay neurospheres were dissociated and plated on Matrigel coated surface in differentiation medium containing basal medium supplemented with N2 (GIBCO) and 1% FBS for five days.

Flow Cytometry:

Article Title: Analysis of transcriptional variability in a large human iPSC library reveals genetic and non-genetic determinants of heterogeneity
Article Snippet: The cells were then cultured for 3 days in DMEM/F12 supplemented with B27 and N2 (Thermo), 5μM CHIR-99021 (Selleckchem) and 25 ng/ml BMP-4 (Peprotech). .. Endothelial differentiation was performed in triplicates and calculated as CD31+/CD144+ cell percentage through flow cytometry.

Microscopy:

Article Title: Differentiation of Multipotent Vascular Stem Cells Contributes to Vascular Diseases
Article Snippet: The surrounding connective tissues and adventitia were dissected away under a dissecting microscope. .. The cells were cultured in DMEM with 10% FBS (Thermo Fisher Scientific Inc.), or in DMEM with 2% CEE (MP Biomedical, Inc.), 1% FBS, 1% N2 (Invitrogen Corp.), 2% B27 (Invitrogen Corp.), 100 nM retinoic acid (RA) (Sigma-Aldrich, Inc.), 50 nM 2-mercaptoethanol (2ME) (Sigma-Aldrich, Inc.), 1% P/S and 20 ng/ml bFGF (R & D Systems, Inc.) (Maintenance medium).

Article Title: LRH-1 mitigates intestinal inflammatory disease by maintaining epithelial homeostasis and cell survival
Article Snippet: Human intestinal organoid culture Endoscopic biopsy samples obtained from the terminal ileum were processed under a dissecting microscope to liberate intestinal crypts using EDTA chelation and mechanical disruption. .. Following polymerization of Matrigel, propagation media (50% LWRN conditioned media from ATCC CRL-3276, supplemented with human EGF (Peprotech), A-83-01 (Tocris), SB202190 (Sigma), Gastrin (Sigma), Nicotinamide (Sigma), B27 (Life Technologies), N2 (Life Technologies), GlutaMax (Life Technologies), and HEPES (Sigma) in F12 Advanced DMEM (Life Technologies)) was added.

Purification:

Article Title: Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture
Article Snippet: Growth factor-free chemically defined medium (gfCDM) was prepared as follows: Iscove’s modified Dulbecco’s medium (IMDM; invitrogen)/Ham’s F12 medium (invitrogen) 1∶1, 1× Chemically-defined lipid concentrate (invitrogen), 450 µM monothioglycerol (Sigma), 5 mg/ml purified bovine serum albumin ( > 99% purified by crystallization; Sigma) and 15 µg/ml apo-transferrin (Sigma). .. On day 5, cell aggregates were transferred to a 10-cm bacterial-grade dish with DMEM/F12 supplemented with N2 (Invitrogen).

Article Title: Role of the JNK Pathway in Varicella-Zoster Virus Lytic Infection and Reactivation
Article Snippet: However, we have employed a novel SOX10-based purification step, which provides a simpler and more reliable and precise method than surface marker staining-based purification or a transgenic Sox10 :: GFP bacterial artificial chromosome (BAC)-based line ( , ). .. Briefly, 500 nM LDN193189 (Abcam) and 10 μM SB431542 (Cayman Chemical) were added for 3 days, followed by additional treatment with Chir 99021 (Tocris Bioscience) for 7 days, with gradual medium change from HES medium to neurobasal medium containing N2 (catalog no. 17502-048; Gibco), B27 (catalog no. 12587-010; Gibco), and 2 mM l -glutamine.

Transgenic Assay:

Article Title: Role of the JNK Pathway in Varicella-Zoster Virus Lytic Infection and Reactivation
Article Snippet: However, we have employed a novel SOX10-based purification step, which provides a simpler and more reliable and precise method than surface marker staining-based purification or a transgenic Sox10 :: GFP bacterial artificial chromosome (BAC)-based line ( , ). .. Briefly, 500 nM LDN193189 (Abcam) and 10 μM SB431542 (Cayman Chemical) were added for 3 days, followed by additional treatment with Chir 99021 (Tocris Bioscience) for 7 days, with gradual medium change from HES medium to neurobasal medium containing N2 (catalog no. 17502-048; Gibco), B27 (catalog no. 12587-010; Gibco), and 2 mM l -glutamine.

Labeling:

Article Title: miR-367 promotes proliferation and stem-like traits in medulloblastoma cells
Article Snippet: Neurosphere formation assay Cells were transfected with miR-367 or non-specific control and plated 24 h later into 96-well low-attachment plates (Corning, New York, NY), at a density of 1 × 103 cells/mL in DMEM/F12 medium containing 20 ng/mL EGF, 20 ng/mL bFGF, 1× B-27 and 1× N2 (Invitrogen). .. For CHLA-01-Med, Daoy and D283-Med cells, 1 × 105 cells were labeled with CD133/1 PE antibody (AC133; Miltenyi Biotec, Cologne, Germany) for 40 min at 4°C.

Staining:

Article Title: Role of the JNK Pathway in Varicella-Zoster Virus Lytic Infection and Reactivation
Article Snippet: However, we have employed a novel SOX10-based purification step, which provides a simpler and more reliable and precise method than surface marker staining-based purification or a transgenic Sox10 :: GFP bacterial artificial chromosome (BAC)-based line ( , ). .. Briefly, 500 nM LDN193189 (Abcam) and 10 μM SB431542 (Cayman Chemical) were added for 3 days, followed by additional treatment with Chir 99021 (Tocris Bioscience) for 7 days, with gradual medium change from HES medium to neurobasal medium containing N2 (catalog no. 17502-048; Gibco), B27 (catalog no. 12587-010; Gibco), and 2 mM l -glutamine.

Plasmid Preparation:

Article Title: Groucho related gene 5 (GRG5) is involved in embryonic and neural stem cell state decisions
Article Snippet: Sequences of the RT primers of the GRG5-TetO-FUW vector are provided in Table . .. For the differentiation assay neurospheres were dissociated and plated on Matrigel coated surface in differentiation medium containing basal medium supplemented with N2 (GIBCO) and 1% FBS for five days.

shRNA:

Article Title: Groucho related gene 5 (GRG5) is involved in embryonic and neural stem cell state decisions
Article Snippet: Neuronal conversion of MEFs 6 × 105 MEFs were seeded on a Matrigel coated 24 well plate and 24 h later coinfected with the lentiviruses ASCL1-TetO-FEW, Myt1l-TetO-FEW, rtTA combined with pLKO.1 or pLKO.1 shRNA against Grg5 respectively (Sigma Aldrich, SHCLNV-NM_010347, TRCN0000097716). .. The next day, the medium was exchanged to iN medium (DMEM/F12 (GIBCO), supplemented with N2 (GIBCO), B27 (GIBCO), 2 mM GlutaMax (GIBCO), 0.05 mg/ml Gentamicin (GIBCO) and 2 μg/ml doxycycline)).

Tube Formation Assay:

Article Title: miR-367 promotes proliferation and stem-like traits in medulloblastoma cells
Article Snippet: .. Neurosphere formation assay Cells were transfected with miR-367 or non-specific control and plated 24 h later into 96-well low-attachment plates (Corning, New York, NY), at a density of 1 × 103 cells/mL in DMEM/F12 medium containing 20 ng/mL EGF, 20 ng/mL bFGF, 1× B-27 and 1× N2 (Invitrogen). ..

Knock-Out:

Article Title: Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture
Article Snippet: Differentiation Medium with KSR was prepared as follows: Glasgow Minimum Essential Medium (GMEM; Invitrogen) supplemented with 5–10% Knockout Serum Replacement (KSR; invitrogen), 1 mM pyruvate (Sigma), 0.1 mM nonessential amino acids (invitrogen), 0.1 mM 2-mercaptoethanol (Sigma). .. On day 5, cell aggregates were transferred to a 10-cm bacterial-grade dish with DMEM/F12 supplemented with N2 (Invitrogen).

Article Title: Role of the JNK Pathway in Varicella-Zoster Virus Lytic Infection and Reactivation
Article Snippet: The HES (WA09/H9; WiCell)-based SOX10::GFP reporter line was cultured with mouse embryonic fibroblasts (ASF-113; Applied Stem Cell) in proliferation medium consisting of Dulbecco's modified Eagle's medium (DMEM)–F-12 (catalog no. 11330-057; Gibco) base medium supplemented with knockout serum (catalog no. 10828-028; Gibco), 2 mM l -glutamine (catalog no. 25030-081; Gibco), MEM nonessential amino acids (catalog no. 11140-050; Gibco), 5.5 mM 2-mercaptoethanol (catalog no. 21985-023; Gibco). .. Briefly, 500 nM LDN193189 (Abcam) and 10 μM SB431542 (Cayman Chemical) were added for 3 days, followed by additional treatment with Chir 99021 (Tocris Bioscience) for 7 days, with gradual medium change from HES medium to neurobasal medium containing N2 (catalog no. 17502-048; Gibco), B27 (catalog no. 12587-010; Gibco), and 2 mM l -glutamine.

Activation Assay:

Article Title: Role of the JNK Pathway in Varicella-Zoster Virus Lytic Infection and Reactivation
Article Snippet: To differentiate SOX10-expressing neural crest stem cells, 6 × 104 HES cells/cm2 on Geltrex (catalog no. A1413302; Gibco) were treated with dual SMAD inhibition and canonical WNT activation, as previously described ( ). .. Briefly, 500 nM LDN193189 (Abcam) and 10 μM SB431542 (Cayman Chemical) were added for 3 days, followed by additional treatment with Chir 99021 (Tocris Bioscience) for 7 days, with gradual medium change from HES medium to neurobasal medium containing N2 (catalog no. 17502-048; Gibco), B27 (catalog no. 12587-010; Gibco), and 2 mM l -glutamine.

BAC Assay:

Article Title: Role of the JNK Pathway in Varicella-Zoster Virus Lytic Infection and Reactivation
Article Snippet: However, we have employed a novel SOX10-based purification step, which provides a simpler and more reliable and precise method than surface marker staining-based purification or a transgenic Sox10 :: GFP bacterial artificial chromosome (BAC)-based line ( , ). .. Briefly, 500 nM LDN193189 (Abcam) and 10 μM SB431542 (Cayman Chemical) were added for 3 days, followed by additional treatment with Chir 99021 (Tocris Bioscience) for 7 days, with gradual medium change from HES medium to neurobasal medium containing N2 (catalog no. 17502-048; Gibco), B27 (catalog no. 12587-010; Gibco), and 2 mM l -glutamine.

Differentiation Assay:

Article Title: Groucho related gene 5 (GRG5) is involved in embryonic and neural stem cell state decisions
Article Snippet: .. For the differentiation assay neurospheres were dissociated and plated on Matrigel coated surface in differentiation medium containing basal medium supplemented with N2 (GIBCO) and 1% FBS for five days. ..

Marker:

Article Title: Role of the JNK Pathway in Varicella-Zoster Virus Lytic Infection and Reactivation
Article Snippet: However, we have employed a novel SOX10-based purification step, which provides a simpler and more reliable and precise method than surface marker staining-based purification or a transgenic Sox10 :: GFP bacterial artificial chromosome (BAC)-based line ( , ). .. Briefly, 500 nM LDN193189 (Abcam) and 10 μM SB431542 (Cayman Chemical) were added for 3 days, followed by additional treatment with Chir 99021 (Tocris Bioscience) for 7 days, with gradual medium change from HES medium to neurobasal medium containing N2 (catalog no. 17502-048; Gibco), B27 (catalog no. 12587-010; Gibco), and 2 mM l -glutamine.

FACS:

Article Title: Role of the JNK Pathway in Varicella-Zoster Virus Lytic Infection and Reactivation
Article Snippet: Briefly, 500 nM LDN193189 (Abcam) and 10 μM SB431542 (Cayman Chemical) were added for 3 days, followed by additional treatment with Chir 99021 (Tocris Bioscience) for 7 days, with gradual medium change from HES medium to neurobasal medium containing N2 (catalog no. 17502-048; Gibco), B27 (catalog no. 12587-010; Gibco), and 2 mM l -glutamine. .. GFP-expressing cells could begin to be identified by day 7, and fluorescence-activated cell sorter (FACS) purification was performed at day 10.

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    Thermo Fisher pegfp n2 plasmid
    Efficiency, viability, and yields of transfection of the <t>pEGFP-N2</t> plasmid into bovine fibroblasts by droplet-EP. A total of 4 × 10 5 bovine earlobe fibroblasts were subjected to droplet- electroporation (EP) at the following settings: 2.0, 3.0, 3.5, or 4.0 kV. Cell viability was determined immediately after charging the cells through droplet-EP. Subsequently, the transfected cells were cultured for 24 hr. After staining the nuclei with Hoechst33342, the transfection efficiency was determined using a laser-scanning microscope. (A) Transfection efficiency was determined based on the proportion of enhanced green fluorescent protein (EGFP)-expressing cells in Hoechst33342-positive cells. (B) A representative image of EGFP-transfected cells. Scale bar=50 µ m. (C) Cell viability was determined using the trypan blue staining method. (D) The yields of transfections were determined by multiplying the efficiency and cell viability obtained at each voltage. “Intact” indicates that droplet-EP was not performed. Values are means ± one standard deviation over three wells. * P
    Pegfp N2 Plasmid, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n2 plasmid/product/Thermo Fisher
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    93
    Thermo Fisher recombinant protein n2 na
    10C4–8E7 MAb specifically decrease the amount of NA of Len/17-MDV in selective passage reassortants pool. (A) Pyrograms of NA gene of clones from selective passage of co-infection between Len/17-MDV (H2N2) and A/Texas/50/2012 (H3N2) in the presence of antibody to HA subtype 2 of A/Japan/305/1957 (H2N2) (Mix 1, representative) or in combination with 10C4–8E7 mAb to <t>N2-NA</t> of Len/17-MDV (Mix 2, representative) and the reference pyrograms of Len/17-MDV and A/Texas/50/2012. The signature nucleotides/peaks of desired A/Texas/50/2012 NA gene in the reassortant are pointed by green arrows, undesired Len/17-MDV NA gene – in red. The nucleotides dispensation order for the sequencing is shown at the bottom. (B) The quantitative assessment of picks height for NA gene was done using “Peak Height Report” of PyroMark Q96 2.5.8 software on pyrograms of RT-PCR products of selective passage RNA (three for each condition). The height of signature peaks of Len/17-MDV NA (C1, T4, T9– red) and the signature peaks of A/Texas/50/2012 NA (C3, T7 - green) was assessed and the average values ± SD for each nucleotide/signature pick are shown.
    Recombinant Protein N2 Na, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher rgf n2
    Phenotypic characterization of immortalized rat portal fibroblastic <t>RGF</t> and <t>RGF-N2</t> cell lines by immunoblot. (A) Immortalized rat PF RGF and RGF-N2 protein samples were analyzed for expression of Gfap and cytokeratins. Housekeeping Gapdh protein was used as loading control, Mz-Cha-1 cell lysate as positive control for cytokeratin expression, and HSC-T6 cell lysate as positive control for Gfap expression. RGF (passage #59, 1 st lane and passage #34, 2 nd lane) and RGF-N2 (passage #52, 1 st lane and passage #25, 2 nd lane) cells do express neither cytokeratin proteins nor Gfap protein (after 45 passages for the latter). (B) Immortalized rat PF RGF and RGF-N2 protein samples were analyzed for expression of αSMA, Elastin, Pdgfrβ and Egfr. RGF and RGF-N2 express all proteins tested. Housekeeping Gapdh protein was used as loading control. kDa , kiloDaltons .
    Rgf N2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher n2 o
    Detection of 15 <t>N2</t> O and 15 N2 in substrate and nutrient solution
    N2 O, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Efficiency, viability, and yields of transfection of the pEGFP-N2 plasmid into bovine fibroblasts by droplet-EP. A total of 4 × 10 5 bovine earlobe fibroblasts were subjected to droplet- electroporation (EP) at the following settings: 2.0, 3.0, 3.5, or 4.0 kV. Cell viability was determined immediately after charging the cells through droplet-EP. Subsequently, the transfected cells were cultured for 24 hr. After staining the nuclei with Hoechst33342, the transfection efficiency was determined using a laser-scanning microscope. (A) Transfection efficiency was determined based on the proportion of enhanced green fluorescent protein (EGFP)-expressing cells in Hoechst33342-positive cells. (B) A representative image of EGFP-transfected cells. Scale bar=50 µ m. (C) Cell viability was determined using the trypan blue staining method. (D) The yields of transfections were determined by multiplying the efficiency and cell viability obtained at each voltage. “Intact” indicates that droplet-EP was not performed. Values are means ± one standard deviation over three wells. * P

    Journal: The Journal of Veterinary Medical Science

    Article Title: Introduction of a plasmid and a protein into bovine and swine cells by water-in-oil droplet electroporation

    doi: 10.1292/jvms.19-0475

    Figure Lengend Snippet: Efficiency, viability, and yields of transfection of the pEGFP-N2 plasmid into bovine fibroblasts by droplet-EP. A total of 4 × 10 5 bovine earlobe fibroblasts were subjected to droplet- electroporation (EP) at the following settings: 2.0, 3.0, 3.5, or 4.0 kV. Cell viability was determined immediately after charging the cells through droplet-EP. Subsequently, the transfected cells were cultured for 24 hr. After staining the nuclei with Hoechst33342, the transfection efficiency was determined using a laser-scanning microscope. (A) Transfection efficiency was determined based on the proportion of enhanced green fluorescent protein (EGFP)-expressing cells in Hoechst33342-positive cells. (B) A representative image of EGFP-transfected cells. Scale bar=50 µ m. (C) Cell viability was determined using the trypan blue staining method. (D) The yields of transfections were determined by multiplying the efficiency and cell viability obtained at each voltage. “Intact” indicates that droplet-EP was not performed. Values are means ± one standard deviation over three wells. * P

    Article Snippet: Briefly, 1.0 × 106 trypsinized bovine or swine fibroblasts and 10 µ g of pEGFP-N2 plasmid in 100 µl of DMEM (Thermo Fisher Scientific) were placed in a 2 mm-gap cuvette (Nepagene) and pulsed using NEPA21.

    Techniques: Transfection, Plasmid Preparation, Electroporation, Cell Culture, Staining, Laser-Scanning Microscopy, Expressing, Standard Deviation

    Efficiency and cytotoxicity of transfection of the pEGFP-N2 plasmid into swine fibroblasts by droplet-electroporation (EP). Swine fibroblasts were isolated from earlobe skin, and subjected to transfection by droplet-EP. (A) Morphology of swine earlobe fibroblasts. Scale bar=50 µ m. (B–D) Transfection efficiency (B), cell viability (C), and yields of transfection (D) were determined (as shown in Fig. 1 ). Values are means ± one standard deviation over three wells. * P

    Journal: The Journal of Veterinary Medical Science

    Article Title: Introduction of a plasmid and a protein into bovine and swine cells by water-in-oil droplet electroporation

    doi: 10.1292/jvms.19-0475

    Figure Lengend Snippet: Efficiency and cytotoxicity of transfection of the pEGFP-N2 plasmid into swine fibroblasts by droplet-electroporation (EP). Swine fibroblasts were isolated from earlobe skin, and subjected to transfection by droplet-EP. (A) Morphology of swine earlobe fibroblasts. Scale bar=50 µ m. (B–D) Transfection efficiency (B), cell viability (C), and yields of transfection (D) were determined (as shown in Fig. 1 ). Values are means ± one standard deviation over three wells. * P

    Article Snippet: Briefly, 1.0 × 106 trypsinized bovine or swine fibroblasts and 10 µ g of pEGFP-N2 plasmid in 100 µl of DMEM (Thermo Fisher Scientific) were placed in a 2 mm-gap cuvette (Nepagene) and pulsed using NEPA21.

    Techniques: Transfection, Plasmid Preparation, Electroporation, Isolation, Standard Deviation

    10C4–8E7 MAb specifically decrease the amount of NA of Len/17-MDV in selective passage reassortants pool. (A) Pyrograms of NA gene of clones from selective passage of co-infection between Len/17-MDV (H2N2) and A/Texas/50/2012 (H3N2) in the presence of antibody to HA subtype 2 of A/Japan/305/1957 (H2N2) (Mix 1, representative) or in combination with 10C4–8E7 mAb to N2-NA of Len/17-MDV (Mix 2, representative) and the reference pyrograms of Len/17-MDV and A/Texas/50/2012. The signature nucleotides/peaks of desired A/Texas/50/2012 NA gene in the reassortant are pointed by green arrows, undesired Len/17-MDV NA gene – in red. The nucleotides dispensation order for the sequencing is shown at the bottom. (B) The quantitative assessment of picks height for NA gene was done using “Peak Height Report” of PyroMark Q96 2.5.8 software on pyrograms of RT-PCR products of selective passage RNA (three for each condition). The height of signature peaks of Len/17-MDV NA (C1, T4, T9– red) and the signature peaks of A/Texas/50/2012 NA (C3, T7 - green) was assessed and the average values ± SD for each nucleotide/signature pick are shown.

    Journal: Virology

    Article Title: Monoclonal antibody against N2 neuraminidase of cold adapted A/Leningrad/134/17/57 (H2N2) enables efficient generation of live attenuated influenza vaccines

    doi: 10.1016/j.virol.2018.07.005

    Figure Lengend Snippet: 10C4–8E7 MAb specifically decrease the amount of NA of Len/17-MDV in selective passage reassortants pool. (A) Pyrograms of NA gene of clones from selective passage of co-infection between Len/17-MDV (H2N2) and A/Texas/50/2012 (H3N2) in the presence of antibody to HA subtype 2 of A/Japan/305/1957 (H2N2) (Mix 1, representative) or in combination with 10C4–8E7 mAb to N2-NA of Len/17-MDV (Mix 2, representative) and the reference pyrograms of Len/17-MDV and A/Texas/50/2012. The signature nucleotides/peaks of desired A/Texas/50/2012 NA gene in the reassortant are pointed by green arrows, undesired Len/17-MDV NA gene – in red. The nucleotides dispensation order for the sequencing is shown at the bottom. (B) The quantitative assessment of picks height for NA gene was done using “Peak Height Report” of PyroMark Q96 2.5.8 software on pyrograms of RT-PCR products of selective passage RNA (three for each condition). The height of signature peaks of Len/17-MDV NA (C1, T4, T9– red) and the signature peaks of A/Texas/50/2012 NA (C3, T7 - green) was assessed and the average values ± SD for each nucleotide/signature pick are shown.

    Article Snippet: Mouse mAb 10C4–8E7 was generated against recombinant protein N2-NA of Len/17-MDV by Pierce Custom Services at Thermo Fisher Scientific and was shown to be specific to Len/17-MDV recN2-NA with no cross reactivity to recN2-NA of A/Perth/09/2009 (H3N2) or recN9-NA of A/Anhui/01/2013 (H7N9).

    Techniques: Clone Assay, Infection, Sequencing, Software, Reverse Transcription Polymerase Chain Reaction

    Phenotypic characterization of immortalized rat portal fibroblastic RGF and RGF-N2 cell lines by immunoblot. (A) Immortalized rat PF RGF and RGF-N2 protein samples were analyzed for expression of Gfap and cytokeratins. Housekeeping Gapdh protein was used as loading control, Mz-Cha-1 cell lysate as positive control for cytokeratin expression, and HSC-T6 cell lysate as positive control for Gfap expression. RGF (passage #59, 1 st lane and passage #34, 2 nd lane) and RGF-N2 (passage #52, 1 st lane and passage #25, 2 nd lane) cells do express neither cytokeratin proteins nor Gfap protein (after 45 passages for the latter). (B) Immortalized rat PF RGF and RGF-N2 protein samples were analyzed for expression of αSMA, Elastin, Pdgfrβ and Egfr. RGF and RGF-N2 express all proteins tested. Housekeeping Gapdh protein was used as loading control. kDa , kiloDaltons .

    Journal: PLoS ONE

    Article Title: Establishment and Characterization of Rat Portal Myofibroblast Cell Lines

    doi: 10.1371/journal.pone.0121161

    Figure Lengend Snippet: Phenotypic characterization of immortalized rat portal fibroblastic RGF and RGF-N2 cell lines by immunoblot. (A) Immortalized rat PF RGF and RGF-N2 protein samples were analyzed for expression of Gfap and cytokeratins. Housekeeping Gapdh protein was used as loading control, Mz-Cha-1 cell lysate as positive control for cytokeratin expression, and HSC-T6 cell lysate as positive control for Gfap expression. RGF (passage #59, 1 st lane and passage #34, 2 nd lane) and RGF-N2 (passage #52, 1 st lane and passage #25, 2 nd lane) cells do express neither cytokeratin proteins nor Gfap protein (after 45 passages for the latter). (B) Immortalized rat PF RGF and RGF-N2 protein samples were analyzed for expression of αSMA, Elastin, Pdgfrβ and Egfr. RGF and RGF-N2 express all proteins tested. Housekeeping Gapdh protein was used as loading control. kDa , kiloDaltons .

    Article Snippet: Confluent RGF, RGF-N2, HSC-T6 and Mz-Cha-1 cells were scraped and lysed with RIPA buffer (Thermo Scientific, Rockford, IL) supplemented with Halt Protease and Phosphatase Inhibitor cocktails (Thermo Scientific) for 5 min.

    Techniques: Expressing, Positive Control

    Phenotypic characterization of immortalized rat portal fibroblastic RGF and RGF-N2 cell lines by immunofluorescence. Immortalized RGF cells were fixed, stained with antibodies to proteins specifically expressed in PF-derived myofibroblasts, and counterstained with DAPI nuclear labeling dye. RGF cells express myofibroblast-specific αSMA and Cd73 proteins, PF-specific elastin and Ntpdase2 proteins, β-actin protein and SV40 antigen. Immortalized RGF-N2 cells were fixed, stained with the same antibodies, and counterstained with DAPI dye, as described above for RGF cells. Like RGF cells, RGF-N2 cells express αSMA, Cd73, Elastin, Ntpdase2, β-actin, and SV40 antigen. 400X magnification .

    Journal: PLoS ONE

    Article Title: Establishment and Characterization of Rat Portal Myofibroblast Cell Lines

    doi: 10.1371/journal.pone.0121161

    Figure Lengend Snippet: Phenotypic characterization of immortalized rat portal fibroblastic RGF and RGF-N2 cell lines by immunofluorescence. Immortalized RGF cells were fixed, stained with antibodies to proteins specifically expressed in PF-derived myofibroblasts, and counterstained with DAPI nuclear labeling dye. RGF cells express myofibroblast-specific αSMA and Cd73 proteins, PF-specific elastin and Ntpdase2 proteins, β-actin protein and SV40 antigen. Immortalized RGF-N2 cells were fixed, stained with the same antibodies, and counterstained with DAPI dye, as described above for RGF cells. Like RGF cells, RGF-N2 cells express αSMA, Cd73, Elastin, Ntpdase2, β-actin, and SV40 antigen. 400X magnification .

    Article Snippet: Confluent RGF, RGF-N2, HSC-T6 and Mz-Cha-1 cells were scraped and lysed with RIPA buffer (Thermo Scientific, Rockford, IL) supplemented with Halt Protease and Phosphatase Inhibitor cocktails (Thermo Scientific) for 5 min.

    Techniques: Immunofluorescence, Staining, Derivative Assay, Labeling

    Co-culture of Mz-Cha-1 cholangiocytes with immortalized rat portal fibroblastic RGF and RGF-N2 cell lines by bromodeoxyuridine incorporation assay. Sub-confluent immortalized human Mz-Cha-1 cholangiocytes were labeled with bromodeoxyuridine reagent for 24 hours (day 1), and co-cultured with RGF and RGF-N2 cells for additional 24 hours (day 2), before assessment of bromodeoxyuridine incorporation. Both RGF ( **** + RGF : M = 50 . 11 , SE = 3 . 899 , vs . alone : M = 100 , SE = 22 . 76 , p˂ . 0001 , n = 4) and RGF-N2 ( ****+RGF-N2 : M = 37 . 64 , SE = 13 . 40 vs . alone : M = 100 , SE = 22 . 76 , p˂ . 0001 , n = 4) cell lines are able to inhibit proliferation of cholangiocytes.

    Journal: PLoS ONE

    Article Title: Establishment and Characterization of Rat Portal Myofibroblast Cell Lines

    doi: 10.1371/journal.pone.0121161

    Figure Lengend Snippet: Co-culture of Mz-Cha-1 cholangiocytes with immortalized rat portal fibroblastic RGF and RGF-N2 cell lines by bromodeoxyuridine incorporation assay. Sub-confluent immortalized human Mz-Cha-1 cholangiocytes were labeled with bromodeoxyuridine reagent for 24 hours (day 1), and co-cultured with RGF and RGF-N2 cells for additional 24 hours (day 2), before assessment of bromodeoxyuridine incorporation. Both RGF ( **** + RGF : M = 50 . 11 , SE = 3 . 899 , vs . alone : M = 100 , SE = 22 . 76 , p˂ . 0001 , n = 4) and RGF-N2 ( ****+RGF-N2 : M = 37 . 64 , SE = 13 . 40 vs . alone : M = 100 , SE = 22 . 76 , p˂ . 0001 , n = 4) cell lines are able to inhibit proliferation of cholangiocytes.

    Article Snippet: Confluent RGF, RGF-N2, HSC-T6 and Mz-Cha-1 cells were scraped and lysed with RIPA buffer (Thermo Scientific, Rockford, IL) supplemented with Halt Protease and Phosphatase Inhibitor cocktails (Thermo Scientific) for 5 min.

    Techniques: Co-Culture Assay, BrdU Incorporation Assay, Labeling, Cell Culture

    Phenotypic characterization of immortalized rat portal fibroblastic RGF and RGF-N2 cell lines by RT-PCR. (A) Cultured primary isolated aPF (8 days) and immortalized rat PF RGF and RGF-N2 cDNA samples were used as templates in PCR reactions with primers specific to transcripts of several fibrogenic genes in liver myofibroblasts (see Table 1 for gene list and accession numbers). RGF and RGF-N2 cell lines express classical αSMA ( Acta2 ), type I collagen α1 ( Col1 α 1 ) liver myofibroblast gene products, along with PF-derived elastin ( Eln ), type XV collagen α1 ( Col15 α 1 ), and Ntpdase2 ( Entpd2 ) myofibroblast markers, also found in primary culture-activated portal myofibroblasts. Both cell lines are devoid of HSC-derived myofibroblastic desmin ( Des ) and lecithin-retinol acyl transferase ( Lrat ) markers. Rat normal liver or brain cDNA was used as positive control, when no PCR amplicon was detected (not shown). Nuclease-free water was used as negative control (not shown). MW , molecular weight; bp , base pairs . (B) Cultured primary isolated aPF (8 days, 2 primary cell isolations) and immortalized rat PF RGF (passages #61 and #64) and RGF-N2 cDNA (passages #54 and #55) samples were used as templates in quantitative PCR reactions with probes specific to transcripts of fibrogenic Acta2 , Eln , Col1 α 1 , Entpd2 , and Nt5e genes in liver myofibroblasts. Housekeeping B2m and Hprt1 genes were separately used as references.

    Journal: PLoS ONE

    Article Title: Establishment and Characterization of Rat Portal Myofibroblast Cell Lines

    doi: 10.1371/journal.pone.0121161

    Figure Lengend Snippet: Phenotypic characterization of immortalized rat portal fibroblastic RGF and RGF-N2 cell lines by RT-PCR. (A) Cultured primary isolated aPF (8 days) and immortalized rat PF RGF and RGF-N2 cDNA samples were used as templates in PCR reactions with primers specific to transcripts of several fibrogenic genes in liver myofibroblasts (see Table 1 for gene list and accession numbers). RGF and RGF-N2 cell lines express classical αSMA ( Acta2 ), type I collagen α1 ( Col1 α 1 ) liver myofibroblast gene products, along with PF-derived elastin ( Eln ), type XV collagen α1 ( Col15 α 1 ), and Ntpdase2 ( Entpd2 ) myofibroblast markers, also found in primary culture-activated portal myofibroblasts. Both cell lines are devoid of HSC-derived myofibroblastic desmin ( Des ) and lecithin-retinol acyl transferase ( Lrat ) markers. Rat normal liver or brain cDNA was used as positive control, when no PCR amplicon was detected (not shown). Nuclease-free water was used as negative control (not shown). MW , molecular weight; bp , base pairs . (B) Cultured primary isolated aPF (8 days, 2 primary cell isolations) and immortalized rat PF RGF (passages #61 and #64) and RGF-N2 cDNA (passages #54 and #55) samples were used as templates in quantitative PCR reactions with probes specific to transcripts of fibrogenic Acta2 , Eln , Col1 α 1 , Entpd2 , and Nt5e genes in liver myofibroblasts. Housekeeping B2m and Hprt1 genes were separately used as references.

    Article Snippet: Confluent RGF, RGF-N2, HSC-T6 and Mz-Cha-1 cells were scraped and lysed with RIPA buffer (Thermo Scientific, Rockford, IL) supplemented with Halt Protease and Phosphatase Inhibitor cocktails (Thermo Scientific) for 5 min.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Isolation, Polymerase Chain Reaction, Derivative Assay, Positive Control, Amplification, Negative Control, Molecular Weight, Real-time Polymerase Chain Reaction

    Plasmid DNA transfer in immortalized rat portal fibroblastic RGF and RGF-N2 cell lines by immunofluorescence. RGF and RGF-N2 cells were transfected with an expression vector encoding monomeric GFP protein cDNA using Fugene6 transfection reagent. Transfected cells were then fixed and counterstained with DAPI dye, before confocal microscopy imaging. Both cell lines exhibit green fluorescence signal, indicative of recombinant GFP protein expression. 200X magnification .

    Journal: PLoS ONE

    Article Title: Establishment and Characterization of Rat Portal Myofibroblast Cell Lines

    doi: 10.1371/journal.pone.0121161

    Figure Lengend Snippet: Plasmid DNA transfer in immortalized rat portal fibroblastic RGF and RGF-N2 cell lines by immunofluorescence. RGF and RGF-N2 cells were transfected with an expression vector encoding monomeric GFP protein cDNA using Fugene6 transfection reagent. Transfected cells were then fixed and counterstained with DAPI dye, before confocal microscopy imaging. Both cell lines exhibit green fluorescence signal, indicative of recombinant GFP protein expression. 200X magnification .

    Article Snippet: Confluent RGF, RGF-N2, HSC-T6 and Mz-Cha-1 cells were scraped and lysed with RIPA buffer (Thermo Scientific, Rockford, IL) supplemented with Halt Protease and Phosphatase Inhibitor cocktails (Thermo Scientific) for 5 min.

    Techniques: Plasmid Preparation, Immunofluorescence, Transfection, Expressing, Confocal Microscopy, Imaging, Fluorescence, Recombinant

    Detection of 15 N2 O and 15 N2 in substrate and nutrient solution

    Journal: Ecology and Evolution

    Article Title: Effects of grass species and grass growth on atmospheric nitrogen deposition to a bog ecosystem surrounded by intensive agricultural land use

    doi: 10.1002/ece3.1534

    Figure Lengend Snippet: Detection of 15 N2 O and 15 N2 in substrate and nutrient solution

    Article Snippet: Soil air and headspace 15 N2 and 15 N2 O samples were measured immediately after sampling using a Stable Isotope Ratio Mass Spectrometer (MAT 253, Thermo Scientific, Bremen, Germany).

    Techniques:

    Detection of 15 N2 O and 15 N2 in substrate and nutrient solution

    Journal: Ecology and Evolution

    Article Title: Effects of grass species and grass growth on atmospheric nitrogen deposition to a bog ecosystem surrounded by intensive agricultural land use

    doi: 10.1002/ece3.1534

    Figure Lengend Snippet: Detection of 15 N2 O and 15 N2 in substrate and nutrient solution

    Article Snippet: Soil air and headspace 15 N2 and 15 N2 O samples were measured immediately after sampling using a Stable Isotope Ratio Mass Spectrometer (MAT 253, Thermo Scientific, Bremen, Germany).

    Techniques: