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  • 93
    Mirus Bio transit 2020
    GFP expression after transfection to Plat-E cells and infection to primary myoblasts Transfection reagents Lipofectamine 2000, Lipofectamine LTX, TransIT-293, <t>TransIT-2020,</t> TransIT-LT1, PolyJet, or LipoJet, were used for pMX-GFP retroviral vector transfection. A. GFP expression in Plat-E cells 2 days after transfection by each transfection reagent, and following infection to primary myoblasts with indicated viral supernatants for 2 days. These phase contrast images show primary myoblasts after infection. C. The number of GFP positive primary myoblasts out of total cells. Data are presented as the mean ± SEM. (n=3)
    Transit 2020, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 93/100, based on 1180 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mirus Bio transit 2020 transfection reagent
    Role of Sp1 binding sites in the promoter activity of the human FCGRT gene. (A) Schematic diagram representing the wild-type promoter construct and the mutant promoter constructs. Mutations are marked with crosses. (B) Summary of the results of luciferase activity. Mutant promoter constructs were generated from the wild-type pGL3-711(WT) subjected to site-directed mutagenesis by the QuikChange Lightning Site-directed Mutagenesis Kit, as described in Materials and Methods. Mutant promoter constructs pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2), pGL3-711(mSp1-3) and pGL3-711(mSp1-1+2+3) contain mutations in the Sp1sequence at positions -641, -635, -641 plus -635, -313, -641 plus -635 plus -313, respectively. Using <t>TransIT-2020</t> <t>transfection</t> reagent, pGL3-basic vector, wild-type pGL3-711(WT) construct and site-directed mutant constructs were transiently cotransfected with the phRG-B plasmid (internal control) into THP-1, Lu 106, Caco-2, and HSkMEC cell lines. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. The transfected THP-1 cells were exposed to PMA (100 ng/ml) for 4 hours prior to harvesting. Transfection efficiency was normalized to the Renilla luciferase activity derived from the phRG-B plasmid and expressed as relative luciferase activity. The data shown are means ± SD of three to six independent experiments performed in duplicate for each construct. The transcriptional activity of the mutant constructs and pGL3-basic is plotted as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. The differences between the wild-type construct and mutant constructs were statistically significant; *P
    Transit 2020 Transfection Reagent, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 92/100, based on 1179 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mirus Bio transit 2020 reagent
    Role of Sp1 binding sites in the promoter activity of the human FCGRT gene. (A) Schematic diagram representing the wild-type promoter construct and the mutant promoter constructs. Mutations are marked with crosses. (B) Summary of the results of luciferase activity. Mutant promoter constructs were generated from the wild-type pGL3-711(WT) subjected to site-directed mutagenesis by the QuikChange Lightning Site-directed Mutagenesis Kit, as described in Materials and Methods. Mutant promoter constructs pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2), pGL3-711(mSp1-3) and pGL3-711(mSp1-1+2+3) contain mutations in the Sp1sequence at positions -641, -635, -641 plus -635, -313, -641 plus -635 plus -313, respectively. Using <t>TransIT-2020</t> <t>transfection</t> reagent, pGL3-basic vector, wild-type pGL3-711(WT) construct and site-directed mutant constructs were transiently cotransfected with the phRG-B plasmid (internal control) into THP-1, Lu 106, Caco-2, and HSkMEC cell lines. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. The transfected THP-1 cells were exposed to PMA (100 ng/ml) for 4 hours prior to harvesting. Transfection efficiency was normalized to the Renilla luciferase activity derived from the phRG-B plasmid and expressed as relative luciferase activity. The data shown are means ± SD of three to six independent experiments performed in duplicate for each construct. The transcriptional activity of the mutant constructs and pGL3-basic is plotted as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. The differences between the wild-type construct and mutant constructs were statistically significant; *P
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    Mirus Bio water soluble cationic lipid transit 2020
    Role of Sp1 binding sites in the promoter activity of the human FCGRT gene. (A) Schematic diagram representing the wild-type promoter construct and the mutant promoter constructs. Mutations are marked with crosses. (B) Summary of the results of luciferase activity. Mutant promoter constructs were generated from the wild-type pGL3-711(WT) subjected to site-directed mutagenesis by the QuikChange Lightning Site-directed Mutagenesis Kit, as described in Materials and Methods. Mutant promoter constructs pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2), pGL3-711(mSp1-3) and pGL3-711(mSp1-1+2+3) contain mutations in the Sp1sequence at positions -641, -635, -641 plus -635, -313, -641 plus -635 plus -313, respectively. Using <t>TransIT-2020</t> <t>transfection</t> reagent, pGL3-basic vector, wild-type pGL3-711(WT) construct and site-directed mutant constructs were transiently cotransfected with the phRG-B plasmid (internal control) into THP-1, Lu 106, Caco-2, and HSkMEC cell lines. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. The transfected THP-1 cells were exposed to PMA (100 ng/ml) for 4 hours prior to harvesting. Transfection efficiency was normalized to the Renilla luciferase activity derived from the phRG-B plasmid and expressed as relative luciferase activity. The data shown are means ± SD of three to six independent experiments performed in duplicate for each construct. The transcriptional activity of the mutant constructs and pGL3-basic is plotted as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. The differences between the wild-type construct and mutant constructs were statistically significant; *P
    Water Soluble Cationic Lipid Transit 2020, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 90/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mirus Bio transit 2020 transfection
    Role of Sp1 binding sites in the promoter activity of the human FCGRT gene. (A) Schematic diagram representing the wild-type promoter construct and the mutant promoter constructs. Mutations are marked with crosses. (B) Summary of the results of luciferase activity. Mutant promoter constructs were generated from the wild-type pGL3-711(WT) subjected to site-directed mutagenesis by the QuikChange Lightning Site-directed Mutagenesis Kit, as described in Materials and Methods. Mutant promoter constructs pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2), pGL3-711(mSp1-3) and pGL3-711(mSp1-1+2+3) contain mutations in the Sp1sequence at positions -641, -635, -641 plus -635, -313, -641 plus -635 plus -313, respectively. Using <t>TransIT-2020</t> <t>transfection</t> reagent, pGL3-basic vector, wild-type pGL3-711(WT) construct and site-directed mutant constructs were transiently cotransfected with the phRG-B plasmid (internal control) into THP-1, Lu 106, Caco-2, and HSkMEC cell lines. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. The transfected THP-1 cells were exposed to PMA (100 ng/ml) for 4 hours prior to harvesting. Transfection efficiency was normalized to the Renilla luciferase activity derived from the phRG-B plasmid and expressed as relative luciferase activity. The data shown are means ± SD of three to six independent experiments performed in duplicate for each construct. The transcriptional activity of the mutant constructs and pGL3-basic is plotted as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. The differences between the wild-type construct and mutant constructs were statistically significant; *P
    Transit 2020 Transfection, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mirus Bio transit 2020 trasnfection reagent
    Role of Sp1 binding sites in the promoter activity of the human FCGRT gene. (A) Schematic diagram representing the wild-type promoter construct and the mutant promoter constructs. Mutations are marked with crosses. (B) Summary of the results of luciferase activity. Mutant promoter constructs were generated from the wild-type pGL3-711(WT) subjected to site-directed mutagenesis by the QuikChange Lightning Site-directed Mutagenesis Kit, as described in Materials and Methods. Mutant promoter constructs pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2), pGL3-711(mSp1-3) and pGL3-711(mSp1-1+2+3) contain mutations in the Sp1sequence at positions -641, -635, -641 plus -635, -313, -641 plus -635 plus -313, respectively. Using <t>TransIT-2020</t> <t>transfection</t> reagent, pGL3-basic vector, wild-type pGL3-711(WT) construct and site-directed mutant constructs were transiently cotransfected with the phRG-B plasmid (internal control) into THP-1, Lu 106, Caco-2, and HSkMEC cell lines. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. The transfected THP-1 cells were exposed to PMA (100 ng/ml) for 4 hours prior to harvesting. Transfection efficiency was normalized to the Renilla luciferase activity derived from the phRG-B plasmid and expressed as relative luciferase activity. The data shown are means ± SD of three to six independent experiments performed in duplicate for each construct. The transcriptional activity of the mutant constructs and pGL3-basic is plotted as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. The differences between the wild-type construct and mutant constructs were statistically significant; *P
    Transit 2020 Trasnfection Reagent, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mirus Bio flow cytometry dna repair reporter assaysusing transit 2020 transfection reagent
    Role of Sp1 binding sites in the promoter activity of the human FCGRT gene. (A) Schematic diagram representing the wild-type promoter construct and the mutant promoter constructs. Mutations are marked with crosses. (B) Summary of the results of luciferase activity. Mutant promoter constructs were generated from the wild-type pGL3-711(WT) subjected to site-directed mutagenesis by the QuikChange Lightning Site-directed Mutagenesis Kit, as described in Materials and Methods. Mutant promoter constructs pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2), pGL3-711(mSp1-3) and pGL3-711(mSp1-1+2+3) contain mutations in the Sp1sequence at positions -641, -635, -641 plus -635, -313, -641 plus -635 plus -313, respectively. Using <t>TransIT-2020</t> <t>transfection</t> reagent, pGL3-basic vector, wild-type pGL3-711(WT) construct and site-directed mutant constructs were transiently cotransfected with the phRG-B plasmid (internal control) into THP-1, Lu 106, Caco-2, and HSkMEC cell lines. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. The transfected THP-1 cells were exposed to PMA (100 ng/ml) for 4 hours prior to harvesting. Transfection efficiency was normalized to the Renilla luciferase activity derived from the phRG-B plasmid and expressed as relative luciferase activity. The data shown are means ± SD of three to six independent experiments performed in duplicate for each construct. The transcriptional activity of the mutant constructs and pGL3-basic is plotted as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. The differences between the wild-type construct and mutant constructs were statistically significant; *P
    Flow Cytometry Dna Repair Reporter Assaysusing Transit 2020 Transfection Reagent, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mirus Bio luciferase assay transit 2020
    Role of Sp1 binding sites in the promoter activity of the human FCGRT gene. (A) Schematic diagram representing the wild-type promoter construct and the mutant promoter constructs. Mutations are marked with crosses. (B) Summary of the results of luciferase activity. Mutant promoter constructs were generated from the wild-type pGL3-711(WT) subjected to site-directed mutagenesis by the QuikChange Lightning Site-directed Mutagenesis Kit, as described in Materials and Methods. Mutant promoter constructs pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2), pGL3-711(mSp1-3) and pGL3-711(mSp1-1+2+3) contain mutations in the Sp1sequence at positions -641, -635, -641 plus -635, -313, -641 plus -635 plus -313, respectively. Using <t>TransIT-2020</t> <t>transfection</t> reagent, pGL3-basic vector, wild-type pGL3-711(WT) construct and site-directed mutant constructs were transiently cotransfected with the phRG-B plasmid (internal control) into THP-1, Lu 106, Caco-2, and HSkMEC cell lines. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. The transfected THP-1 cells were exposed to PMA (100 ng/ml) for 4 hours prior to harvesting. Transfection efficiency was normalized to the Renilla luciferase activity derived from the phRG-B plasmid and expressed as relative luciferase activity. The data shown are means ± SD of three to six independent experiments performed in duplicate for each construct. The transcriptional activity of the mutant constructs and pGL3-basic is plotted as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. The differences between the wild-type construct and mutant constructs were statistically significant; *P
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    Mirus Bio serum compatible broad spectrum transfection reagent transit 2020
    Role of Sp1 binding sites in the promoter activity of the human FCGRT gene. (A) Schematic diagram representing the wild-type promoter construct and the mutant promoter constructs. Mutations are marked with crosses. (B) Summary of the results of luciferase activity. Mutant promoter constructs were generated from the wild-type pGL3-711(WT) subjected to site-directed mutagenesis by the QuikChange Lightning Site-directed Mutagenesis Kit, as described in Materials and Methods. Mutant promoter constructs pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2), pGL3-711(mSp1-3) and pGL3-711(mSp1-1+2+3) contain mutations in the Sp1sequence at positions -641, -635, -641 plus -635, -313, -641 plus -635 plus -313, respectively. Using <t>TransIT-2020</t> <t>transfection</t> reagent, pGL3-basic vector, wild-type pGL3-711(WT) construct and site-directed mutant constructs were transiently cotransfected with the phRG-B plasmid (internal control) into THP-1, Lu 106, Caco-2, and HSkMEC cell lines. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. The transfected THP-1 cells were exposed to PMA (100 ng/ml) for 4 hours prior to harvesting. Transfection efficiency was normalized to the Renilla luciferase activity derived from the phRG-B plasmid and expressed as relative luciferase activity. The data shown are means ± SD of three to six independent experiments performed in duplicate for each construct. The transcriptional activity of the mutant constructs and pGL3-basic is plotted as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. The differences between the wild-type construct and mutant constructs were statistically significant; *P
    Serum Compatible Broad Spectrum Transfection Reagent Transit 2020, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mirus Bio 293t cells employing transit 2020
    Role of Sp1 binding sites in the promoter activity of the human FCGRT gene. (A) Schematic diagram representing the wild-type promoter construct and the mutant promoter constructs. Mutations are marked with crosses. (B) Summary of the results of luciferase activity. Mutant promoter constructs were generated from the wild-type pGL3-711(WT) subjected to site-directed mutagenesis by the QuikChange Lightning Site-directed Mutagenesis Kit, as described in Materials and Methods. Mutant promoter constructs pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2), pGL3-711(mSp1-3) and pGL3-711(mSp1-1+2+3) contain mutations in the Sp1sequence at positions -641, -635, -641 plus -635, -313, -641 plus -635 plus -313, respectively. Using <t>TransIT-2020</t> <t>transfection</t> reagent, pGL3-basic vector, wild-type pGL3-711(WT) construct and site-directed mutant constructs were transiently cotransfected with the phRG-B plasmid (internal control) into THP-1, Lu 106, Caco-2, and HSkMEC cell lines. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. The transfected THP-1 cells were exposed to PMA (100 ng/ml) for 4 hours prior to harvesting. Transfection efficiency was normalized to the Renilla luciferase activity derived from the phRG-B plasmid and expressed as relative luciferase activity. The data shown are means ± SD of three to six independent experiments performed in duplicate for each construct. The transcriptional activity of the mutant constructs and pGL3-basic is plotted as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. The differences between the wild-type construct and mutant constructs were statistically significant; *P
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    Mirus Bio mirus transit 2020 kit
    Role of Sp1 binding sites in the promoter activity of the human FCGRT gene. (A) Schematic diagram representing the wild-type promoter construct and the mutant promoter constructs. Mutations are marked with crosses. (B) Summary of the results of luciferase activity. Mutant promoter constructs were generated from the wild-type pGL3-711(WT) subjected to site-directed mutagenesis by the QuikChange Lightning Site-directed Mutagenesis Kit, as described in Materials and Methods. Mutant promoter constructs pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2), pGL3-711(mSp1-3) and pGL3-711(mSp1-1+2+3) contain mutations in the Sp1sequence at positions -641, -635, -641 plus -635, -313, -641 plus -635 plus -313, respectively. Using <t>TransIT-2020</t> <t>transfection</t> reagent, pGL3-basic vector, wild-type pGL3-711(WT) construct and site-directed mutant constructs were transiently cotransfected with the phRG-B plasmid (internal control) into THP-1, Lu 106, Caco-2, and HSkMEC cell lines. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. The transfected THP-1 cells were exposed to PMA (100 ng/ml) for 4 hours prior to harvesting. Transfection efficiency was normalized to the Renilla luciferase activity derived from the phRG-B plasmid and expressed as relative luciferase activity. The data shown are means ± SD of three to six independent experiments performed in duplicate for each construct. The transcriptional activity of the mutant constructs and pGL3-basic is plotted as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. The differences between the wild-type construct and mutant constructs were statistically significant; *P
    Mirus Transit 2020 Kit, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 88/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    allele biotechnology transit 2020 transfection reagent
    Role of Sp1 binding sites in the promoter activity of the human FCGRT gene. (A) Schematic diagram representing the wild-type promoter construct and the mutant promoter constructs. Mutations are marked with crosses. (B) Summary of the results of luciferase activity. Mutant promoter constructs were generated from the wild-type pGL3-711(WT) subjected to site-directed mutagenesis by the QuikChange Lightning Site-directed Mutagenesis Kit, as described in Materials and Methods. Mutant promoter constructs pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2), pGL3-711(mSp1-3) and pGL3-711(mSp1-1+2+3) contain mutations in the Sp1sequence at positions -641, -635, -641 plus -635, -313, -641 plus -635 plus -313, respectively. Using <t>TransIT-2020</t> <t>transfection</t> reagent, pGL3-basic vector, wild-type pGL3-711(WT) construct and site-directed mutant constructs were transiently cotransfected with the phRG-B plasmid (internal control) into THP-1, Lu 106, Caco-2, and HSkMEC cell lines. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. The transfected THP-1 cells were exposed to PMA (100 ng/ml) for 4 hours prior to harvesting. Transfection efficiency was normalized to the Renilla luciferase activity derived from the phRG-B plasmid and expressed as relative luciferase activity. The data shown are means ± SD of three to six independent experiments performed in duplicate for each construct. The transcriptional activity of the mutant constructs and pGL3-basic is plotted as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. The differences between the wild-type construct and mutant constructs were statistically significant; *P
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    Avantor transit 2020 transfection reagent
    Role of Sp1 binding sites in the promoter activity of the human FCGRT gene. (A) Schematic diagram representing the wild-type promoter construct and the mutant promoter constructs. Mutations are marked with crosses. (B) Summary of the results of luciferase activity. Mutant promoter constructs were generated from the wild-type pGL3-711(WT) subjected to site-directed mutagenesis by the QuikChange Lightning Site-directed Mutagenesis Kit, as described in Materials and Methods. Mutant promoter constructs pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2), pGL3-711(mSp1-3) and pGL3-711(mSp1-1+2+3) contain mutations in the Sp1sequence at positions -641, -635, -641 plus -635, -313, -641 plus -635 plus -313, respectively. Using <t>TransIT-2020</t> <t>transfection</t> reagent, pGL3-basic vector, wild-type pGL3-711(WT) construct and site-directed mutant constructs were transiently cotransfected with the phRG-B plasmid (internal control) into THP-1, Lu 106, Caco-2, and HSkMEC cell lines. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. The transfected THP-1 cells were exposed to PMA (100 ng/ml) for 4 hours prior to harvesting. Transfection efficiency was normalized to the Renilla luciferase activity derived from the phRG-B plasmid and expressed as relative luciferase activity. The data shown are means ± SD of three to six independent experiments performed in duplicate for each construct. The transcriptional activity of the mutant constructs and pGL3-basic is plotted as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. The differences between the wild-type construct and mutant constructs were statistically significant; *P
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    GFP expression after transfection to Plat-E cells and infection to primary myoblasts Transfection reagents Lipofectamine 2000, Lipofectamine LTX, TransIT-293, TransIT-2020, TransIT-LT1, PolyJet, or LipoJet, were used for pMX-GFP retroviral vector transfection. A. GFP expression in Plat-E cells 2 days after transfection by each transfection reagent, and following infection to primary myoblasts with indicated viral supernatants for 2 days. These phase contrast images show primary myoblasts after infection. C. The number of GFP positive primary myoblasts out of total cells. Data are presented as the mean ± SEM. (n=3)

    Journal: BioTechniques

    Article Title: Spin infection for efficient gene delivery in muscle stem cells for intramuscular cell transplantation

    doi: 10.2144/000114576

    Figure Lengend Snippet: GFP expression after transfection to Plat-E cells and infection to primary myoblasts Transfection reagents Lipofectamine 2000, Lipofectamine LTX, TransIT-293, TransIT-2020, TransIT-LT1, PolyJet, or LipoJet, were used for pMX-GFP retroviral vector transfection. A. GFP expression in Plat-E cells 2 days after transfection by each transfection reagent, and following infection to primary myoblasts with indicated viral supernatants for 2 days. These phase contrast images show primary myoblasts after infection. C. The number of GFP positive primary myoblasts out of total cells. Data are presented as the mean ± SEM. (n=3)

    Article Snippet: Various transfection reagents were used, such as: Lipofectamine (Thermo Fisher Scientific, Waltham, MA, USA), Lipofectamine 2000 (Thermo Fisher Scientific), Lipofectamine LTX (Thermo Fisher Scientific), TransIT-293(Mirus Bio LLC, Madison, WI, USA), TransIT-2020 (Mirus Bio LLC), TransIT-LT1 (Mirus Bio LLC), PolyJet (SignaGen Laboratories, Rockville, MD, USA) and LipoJet (SignaGen Laboratories).

    Techniques: Expressing, Transfection, Infection, Plasmid Preparation

    Role of Sp1 binding sites in the promoter activity of the human FCGRT gene. (A) Schematic diagram representing the wild-type promoter construct and the mutant promoter constructs. Mutations are marked with crosses. (B) Summary of the results of luciferase activity. Mutant promoter constructs were generated from the wild-type pGL3-711(WT) subjected to site-directed mutagenesis by the QuikChange Lightning Site-directed Mutagenesis Kit, as described in Materials and Methods. Mutant promoter constructs pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2), pGL3-711(mSp1-3) and pGL3-711(mSp1-1+2+3) contain mutations in the Sp1sequence at positions -641, -635, -641 plus -635, -313, -641 plus -635 plus -313, respectively. Using TransIT-2020 transfection reagent, pGL3-basic vector, wild-type pGL3-711(WT) construct and site-directed mutant constructs were transiently cotransfected with the phRG-B plasmid (internal control) into THP-1, Lu 106, Caco-2, and HSkMEC cell lines. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. The transfected THP-1 cells were exposed to PMA (100 ng/ml) for 4 hours prior to harvesting. Transfection efficiency was normalized to the Renilla luciferase activity derived from the phRG-B plasmid and expressed as relative luciferase activity. The data shown are means ± SD of three to six independent experiments performed in duplicate for each construct. The transcriptional activity of the mutant constructs and pGL3-basic is plotted as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. The differences between the wild-type construct and mutant constructs were statistically significant; *P

    Journal: PLoS ONE

    Article Title: Analysis of Response Elements Involved in the Regulation of the Human Neonatal Fc Receptor Gene (FCGRT)

    doi: 10.1371/journal.pone.0135141

    Figure Lengend Snippet: Role of Sp1 binding sites in the promoter activity of the human FCGRT gene. (A) Schematic diagram representing the wild-type promoter construct and the mutant promoter constructs. Mutations are marked with crosses. (B) Summary of the results of luciferase activity. Mutant promoter constructs were generated from the wild-type pGL3-711(WT) subjected to site-directed mutagenesis by the QuikChange Lightning Site-directed Mutagenesis Kit, as described in Materials and Methods. Mutant promoter constructs pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2), pGL3-711(mSp1-3) and pGL3-711(mSp1-1+2+3) contain mutations in the Sp1sequence at positions -641, -635, -641 plus -635, -313, -641 plus -635 plus -313, respectively. Using TransIT-2020 transfection reagent, pGL3-basic vector, wild-type pGL3-711(WT) construct and site-directed mutant constructs were transiently cotransfected with the phRG-B plasmid (internal control) into THP-1, Lu 106, Caco-2, and HSkMEC cell lines. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. The transfected THP-1 cells were exposed to PMA (100 ng/ml) for 4 hours prior to harvesting. Transfection efficiency was normalized to the Renilla luciferase activity derived from the phRG-B plasmid and expressed as relative luciferase activity. The data shown are means ± SD of three to six independent experiments performed in duplicate for each construct. The transcriptional activity of the mutant constructs and pGL3-basic is plotted as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. The differences between the wild-type construct and mutant constructs were statistically significant; *P

    Article Snippet: TransIT-2020 transfection reagent was purchased from Mirus Bio (USA).

    Techniques: Binding Assay, Activity Assay, Construct, Mutagenesis, Luciferase, Generated, Transfection, Plasmid Preparation, Derivative Assay