Synthesized:Article Title: Identification and functional characterization of a new IL-1 family member, IL-1Fm2, in most evolutionarily advanced fish.
Article Snippet: .. Briefly, the open reading frame (ORF) of IL-1Fm2 encoding the putative mature protein (residues 118–270) was synthesized, cloned in vector E3, produced as an N-terminal 6 His fusion protein in E. coli, obtained from inclusion bodies with 6M guanidine hydrochloride and purified by at St Petersburg State University on December 31, 2013ini.sagepub.comDownloaded from immobilized-metal affinity chromatography using Ni-HiTrap columns (GenScript, Piscataway, NJ, USA). .. Seabream head–kidney leukocytes (bone marrow equivalent of fish) obtained and cultured as described elsewhere20 were stimulated for 4 h at 23 C with 50 mg/ ml phenol-extracted genomic DNA from V. anguillarum ATCC19264 cells (VaDNA).21 Some experiments were conducted using purified acidophilic granulocytes obtained by magnetic-activated cell sorting using a mAb specific to these cells21 and macrophage monolayers.22 Macrophages and acidophilic granulocytes were stimulated for 0.5 to 24 h at 23 C with 50 mg/ml VaDNA, 1 ng/ml flagellin (Invivogen, Madrid, Spain), 25 mg/ml poly I:C (Invivogen, San Diego, CA, USA), 10 mg/ml LPS from E. coli (EcLPS; Sigma-Aldrich, Madrid, Spain), 10 mg/ml Porphyromonas gingivalis LPS (PgLPS; Invivogen) or 10 mg/ml muramyldipeptide (MDP; Sigma-Aldrich).21,23 To examine the effect of recombinant seabream IL-1Fm2 on the macrophages and acidophilic granulocytes, they were stimulated for 3 (macrophages) or 16 h (acidophilic granulocytes) at 23 C with 50 mg/ml VaDNA and 10 ng/ml recombinant seabream IL-1Fm2. cDNA was produced from the cells, and expression of IL-1b, IL-1Fm2, TNF-a, IL-8 and IL-10 was then analyzed using RT-qPCR.
Clone Assay:Article Title: Identification and functional characterization of a new IL-1 family member, IL-1Fm2, in most evolutionarily advanced fish.
Article Snippet: .. Briefly, the open reading frame (ORF) of IL-1Fm2 encoding the putative mature protein (residues 118–270) was synthesized, cloned in vector E3, produced as an N-terminal 6 His fusion protein in E. coli, obtained from inclusion bodies with 6M guanidine hydrochloride and purified by at St Petersburg State University on December 31, 2013ini.sagepub.comDownloaded from immobilized-metal affinity chromatography using Ni-HiTrap columns (GenScript, Piscataway, NJ, USA). .. Seabream head–kidney leukocytes (bone marrow equivalent of fish) obtained and cultured as described elsewhere20 were stimulated for 4 h at 23 C with 50 mg/ ml phenol-extracted genomic DNA from V. anguillarum ATCC19264 cells (VaDNA).21 Some experiments were conducted using purified acidophilic granulocytes obtained by magnetic-activated cell sorting using a mAb specific to these cells21 and macrophage monolayers.22 Macrophages and acidophilic granulocytes were stimulated for 0.5 to 24 h at 23 C with 50 mg/ml VaDNA, 1 ng/ml flagellin (Invivogen, Madrid, Spain), 25 mg/ml poly I:C (Invivogen, San Diego, CA, USA), 10 mg/ml LPS from E. coli (EcLPS; Sigma-Aldrich, Madrid, Spain), 10 mg/ml Porphyromonas gingivalis LPS (PgLPS; Invivogen) or 10 mg/ml muramyldipeptide (MDP; Sigma-Aldrich).21,23 To examine the effect of recombinant seabream IL-1Fm2 on the macrophages and acidophilic granulocytes, they were stimulated for 3 (macrophages) or 16 h (acidophilic granulocytes) at 23 C with 50 mg/ml VaDNA and 10 ng/ml recombinant seabream IL-1Fm2. cDNA was produced from the cells, and expression of IL-1b, IL-1Fm2, TNF-a, IL-8 and IL-10 was then analyzed using RT-qPCR.
Plasmid Preparation:Article Title: Identification and functional characterization of a new IL-1 family member, IL-1Fm2, in most evolutionarily advanced fish.
Article Snippet: .. Briefly, the open reading frame (ORF) of IL-1Fm2 encoding the putative mature protein (residues 118–270) was synthesized, cloned in vector E3, produced as an N-terminal 6 His fusion protein in E. coli, obtained from inclusion bodies with 6M guanidine hydrochloride and purified by at St Petersburg State University on December 31, 2013ini.sagepub.comDownloaded from immobilized-metal affinity chromatography using Ni-HiTrap columns (GenScript, Piscataway, NJ, USA). .. Seabream head–kidney leukocytes (bone marrow equivalent of fish) obtained and cultured as described elsewhere20 were stimulated for 4 h at 23 C with 50 mg/ ml phenol-extracted genomic DNA from V. anguillarum ATCC19264 cells (VaDNA).21 Some experiments were conducted using purified acidophilic granulocytes obtained by magnetic-activated cell sorting using a mAb specific to these cells21 and macrophage monolayers.22 Macrophages and acidophilic granulocytes were stimulated for 0.5 to 24 h at 23 C with 50 mg/ml VaDNA, 1 ng/ml flagellin (Invivogen, Madrid, Spain), 25 mg/ml poly I:C (Invivogen, San Diego, CA, USA), 10 mg/ml LPS from E. coli (EcLPS; Sigma-Aldrich, Madrid, Spain), 10 mg/ml Porphyromonas gingivalis LPS (PgLPS; Invivogen) or 10 mg/ml muramyldipeptide (MDP; Sigma-Aldrich).21,23 To examine the effect of recombinant seabream IL-1Fm2 on the macrophages and acidophilic granulocytes, they were stimulated for 3 (macrophages) or 16 h (acidophilic granulocytes) at 23 C with 50 mg/ml VaDNA and 10 ng/ml recombinant seabream IL-1Fm2. cDNA was produced from the cells, and expression of IL-1b, IL-1Fm2, TNF-a, IL-8 and IL-10 was then analyzed using RT-qPCR.
Produced:Article Title: Identification and functional characterization of a new IL-1 family member, IL-1Fm2, in most evolutionarily advanced fish.
Article Snippet: .. Briefly, the open reading frame (ORF) of IL-1Fm2 encoding the putative mature protein (residues 118–270) was synthesized, cloned in vector E3, produced as an N-terminal 6 His fusion protein in E. coli, obtained from inclusion bodies with 6M guanidine hydrochloride and purified by at St Petersburg State University on December 31, 2013ini.sagepub.comDownloaded from immobilized-metal affinity chromatography using Ni-HiTrap columns (GenScript, Piscataway, NJ, USA). .. Seabream head–kidney leukocytes (bone marrow equivalent of fish) obtained and cultured as described elsewhere20 were stimulated for 4 h at 23 C with 50 mg/ ml phenol-extracted genomic DNA from V. anguillarum ATCC19264 cells (VaDNA).21 Some experiments were conducted using purified acidophilic granulocytes obtained by magnetic-activated cell sorting using a mAb specific to these cells21 and macrophage monolayers.22 Macrophages and acidophilic granulocytes were stimulated for 0.5 to 24 h at 23 C with 50 mg/ml VaDNA, 1 ng/ml flagellin (Invivogen, Madrid, Spain), 25 mg/ml poly I:C (Invivogen, San Diego, CA, USA), 10 mg/ml LPS from E. coli (EcLPS; Sigma-Aldrich, Madrid, Spain), 10 mg/ml Porphyromonas gingivalis LPS (PgLPS; Invivogen) or 10 mg/ml muramyldipeptide (MDP; Sigma-Aldrich).21,23 To examine the effect of recombinant seabream IL-1Fm2 on the macrophages and acidophilic granulocytes, they were stimulated for 3 (macrophages) or 16 h (acidophilic granulocytes) at 23 C with 50 mg/ml VaDNA and 10 ng/ml recombinant seabream IL-1Fm2. cDNA was produced from the cells, and expression of IL-1b, IL-1Fm2, TNF-a, IL-8 and IL-10 was then analyzed using RT-qPCR.
Purification:Article Title: Identification and functional characterization of a new IL-1 family member, IL-1Fm2, in most evolutionarily advanced fish.
Article Snippet: .. Briefly, the open reading frame (ORF) of IL-1Fm2 encoding the putative mature protein (residues 118–270) was synthesized, cloned in vector E3, produced as an N-terminal 6 His fusion protein in E. coli, obtained from inclusion bodies with 6M guanidine hydrochloride and purified by at St Petersburg State University on December 31, 2013ini.sagepub.comDownloaded from immobilized-metal affinity chromatography using Ni-HiTrap columns (GenScript, Piscataway, NJ, USA). .. Seabream head–kidney leukocytes (bone marrow equivalent of fish) obtained and cultured as described elsewhere20 were stimulated for 4 h at 23 C with 50 mg/ ml phenol-extracted genomic DNA from V. anguillarum ATCC19264 cells (VaDNA).21 Some experiments were conducted using purified acidophilic granulocytes obtained by magnetic-activated cell sorting using a mAb specific to these cells21 and macrophage monolayers.22 Macrophages and acidophilic granulocytes were stimulated for 0.5 to 24 h at 23 C with 50 mg/ml VaDNA, 1 ng/ml flagellin (Invivogen, Madrid, Spain), 25 mg/ml poly I:C (Invivogen, San Diego, CA, USA), 10 mg/ml LPS from E. coli (EcLPS; Sigma-Aldrich, Madrid, Spain), 10 mg/ml Porphyromonas gingivalis LPS (PgLPS; Invivogen) or 10 mg/ml muramyldipeptide (MDP; Sigma-Aldrich).21,23 To examine the effect of recombinant seabream IL-1Fm2 on the macrophages and acidophilic granulocytes, they were stimulated for 3 (macrophages) or 16 h (acidophilic granulocytes) at 23 C with 50 mg/ml VaDNA and 10 ng/ml recombinant seabream IL-1Fm2. cDNA was produced from the cells, and expression of IL-1b, IL-1Fm2, TNF-a, IL-8 and IL-10 was then analyzed using RT-qPCR.
Affinity Chromatography:Article Title: Identification and functional characterization of a new IL-1 family member, IL-1Fm2, in most evolutionarily advanced fish.
Article Snippet: .. Briefly, the open reading frame (ORF) of IL-1Fm2 encoding the putative mature protein (residues 118–270) was synthesized, cloned in vector E3, produced as an N-terminal 6 His fusion protein in E. coli, obtained from inclusion bodies with 6M guanidine hydrochloride and purified by at St Petersburg State University on December 31, 2013ini.sagepub.comDownloaded from immobilized-metal affinity chromatography using Ni-HiTrap columns (GenScript, Piscataway, NJ, USA). .. Seabream head–kidney leukocytes (bone marrow equivalent of fish) obtained and cultured as described elsewhere20 were stimulated for 4 h at 23 C with 50 mg/ ml phenol-extracted genomic DNA from V. anguillarum ATCC19264 cells (VaDNA).21 Some experiments were conducted using purified acidophilic granulocytes obtained by magnetic-activated cell sorting using a mAb specific to these cells21 and macrophage monolayers.22 Macrophages and acidophilic granulocytes were stimulated for 0.5 to 24 h at 23 C with 50 mg/ml VaDNA, 1 ng/ml flagellin (Invivogen, Madrid, Spain), 25 mg/ml poly I:C (Invivogen, San Diego, CA, USA), 10 mg/ml LPS from E. coli (EcLPS; Sigma-Aldrich, Madrid, Spain), 10 mg/ml Porphyromonas gingivalis LPS (PgLPS; Invivogen) or 10 mg/ml muramyldipeptide (MDP; Sigma-Aldrich).21,23 To examine the effect of recombinant seabream IL-1Fm2 on the macrophages and acidophilic granulocytes, they were stimulated for 3 (macrophages) or 16 h (acidophilic granulocytes) at 23 C with 50 mg/ml VaDNA and 10 ng/ml recombinant seabream IL-1Fm2. cDNA was produced from the cells, and expression of IL-1b, IL-1Fm2, TNF-a, IL-8 and IL-10 was then analyzed using RT-qPCR.
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