bsc 40  (ATCC)


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    ATCC bsc 40
    A) Scheme of construction. The plasmid transfer vector pGem-RG-B19R wm was obtained by sequential cloning of selectable markers dsRed2 and rsGFP and B19R recombination flanking sequences into the plasmid pGem-7Zf. For the generation of NYVAC-C-ΔB19R, <t>BSC-40</t> cells were infected at an MOI of 0.01 with NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm. 48 hours post-infection, the cells were harvested, lysed by freeze-thaw cycling, sonicated and used for recombinant virus screening. The deletion mutant was selected from progeny virus by consecutive rounds of plaque purification in BSC-40 cells during which plaques were screened for Red2/GFP fluorescence. B) PCR analysis. Viral DNA was extracted from BSC-40 cells mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. Primers LFB19R-AatII-F and LFB19R-BamHI-R spanning B19R flanking regions were used for PCR analysis of B19R locus. C) Expression of HIV-1 antigens gp120 and GPN. BSC-40 cells were mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At 48 hours post-infection, cells were lysed in Laemmli buffer, cells extracts were fractionated by 10% SDS-PAGE and analyzed by Western blot using rabbit polyclonal anti-gp120 antibody or polyclonal anti-gag p24 serum. D) Replication in CEF cells. CEF cells were infected at an MOI of 0.01 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At different times post-infection (hpi, 0, 24, 48 and 72 hours), cells were harvested, freeze-thawed three times and sonicated. Virus titers in cell lysates were determined by crystal violet staining in BSC-40 cells.
    Bsc 40, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Improved NYVAC-Based Vaccine Vectors"

    Article Title: Improved NYVAC-Based Vaccine Vectors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025674

    A) Scheme of construction. The plasmid transfer vector pGem-RG-B19R wm was obtained by sequential cloning of selectable markers dsRed2 and rsGFP and B19R recombination flanking sequences into the plasmid pGem-7Zf. For the generation of NYVAC-C-ΔB19R, BSC-40 cells were infected at an MOI of 0.01 with NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm. 48 hours post-infection, the cells were harvested, lysed by freeze-thaw cycling, sonicated and used for recombinant virus screening. The deletion mutant was selected from progeny virus by consecutive rounds of plaque purification in BSC-40 cells during which plaques were screened for Red2/GFP fluorescence. B) PCR analysis. Viral DNA was extracted from BSC-40 cells mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. Primers LFB19R-AatII-F and LFB19R-BamHI-R spanning B19R flanking regions were used for PCR analysis of B19R locus. C) Expression of HIV-1 antigens gp120 and GPN. BSC-40 cells were mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At 48 hours post-infection, cells were lysed in Laemmli buffer, cells extracts were fractionated by 10% SDS-PAGE and analyzed by Western blot using rabbit polyclonal anti-gp120 antibody or polyclonal anti-gag p24 serum. D) Replication in CEF cells. CEF cells were infected at an MOI of 0.01 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At different times post-infection (hpi, 0, 24, 48 and 72 hours), cells were harvested, freeze-thawed three times and sonicated. Virus titers in cell lysates were determined by crystal violet staining in BSC-40 cells.
    Figure Legend Snippet: A) Scheme of construction. The plasmid transfer vector pGem-RG-B19R wm was obtained by sequential cloning of selectable markers dsRed2 and rsGFP and B19R recombination flanking sequences into the plasmid pGem-7Zf. For the generation of NYVAC-C-ΔB19R, BSC-40 cells were infected at an MOI of 0.01 with NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm. 48 hours post-infection, the cells were harvested, lysed by freeze-thaw cycling, sonicated and used for recombinant virus screening. The deletion mutant was selected from progeny virus by consecutive rounds of plaque purification in BSC-40 cells during which plaques were screened for Red2/GFP fluorescence. B) PCR analysis. Viral DNA was extracted from BSC-40 cells mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. Primers LFB19R-AatII-F and LFB19R-BamHI-R spanning B19R flanking regions were used for PCR analysis of B19R locus. C) Expression of HIV-1 antigens gp120 and GPN. BSC-40 cells were mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At 48 hours post-infection, cells were lysed in Laemmli buffer, cells extracts were fractionated by 10% SDS-PAGE and analyzed by Western blot using rabbit polyclonal anti-gp120 antibody or polyclonal anti-gag p24 serum. D) Replication in CEF cells. CEF cells were infected at an MOI of 0.01 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At different times post-infection (hpi, 0, 24, 48 and 72 hours), cells were harvested, freeze-thawed three times and sonicated. Virus titers in cell lysates were determined by crystal violet staining in BSC-40 cells.

    Techniques Used: Plasmid Preparation, Clone Assay, Infection, Transfection, Sonication, Recombinant, Mutagenesis, Purification, Fluorescence, Expressing, SDS Page, Western Blot, Staining

    Five groups of mice (n = 6) received an IP inoculation of 2×10 7 PFU/mouse of WR (TK − ), NYVAC-C, NYVAC-C-KC, NYVAC-C-ΔB19R or NYVAC-C-KC-ΔB19R. At different times post-inoculation (24, 48 and 72 hours) animals were sacrificed and different mouse tissues (peritoneal cells, ovaries, livers, spleens and draining lymph nodes) were harvested and processed for titration as described in . The production of infectious virus was determined by plaque assay in BSC-40 cells. The virus titer was expressed as Plaque Forming Units (pfu) per gram of protein.
    Figure Legend Snippet: Five groups of mice (n = 6) received an IP inoculation of 2×10 7 PFU/mouse of WR (TK − ), NYVAC-C, NYVAC-C-KC, NYVAC-C-ΔB19R or NYVAC-C-KC-ΔB19R. At different times post-inoculation (24, 48 and 72 hours) animals were sacrificed and different mouse tissues (peritoneal cells, ovaries, livers, spleens and draining lymph nodes) were harvested and processed for titration as described in . The production of infectious virus was determined by plaque assay in BSC-40 cells. The virus titer was expressed as Plaque Forming Units (pfu) per gram of protein.

    Techniques Used: Titration, Plaque Assay

    bsc 40  (ATCC)


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    ATCC bsc 40
    A) Scheme of construction. The plasmid transfer vector pGem-RG-B19R wm was obtained by sequential cloning of selectable markers dsRed2 and rsGFP and B19R recombination flanking sequences into the plasmid pGem-7Zf. For the generation of NYVAC-C-ΔB19R, <t>BSC-40</t> cells were infected at an MOI of 0.01 with NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm. 48 hours post-infection, the cells were harvested, lysed by freeze-thaw cycling, sonicated and used for recombinant virus screening. The deletion mutant was selected from progeny virus by consecutive rounds of plaque purification in BSC-40 cells during which plaques were screened for Red2/GFP fluorescence. B) PCR analysis. Viral DNA was extracted from BSC-40 cells mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. Primers LFB19R-AatII-F and LFB19R-BamHI-R spanning B19R flanking regions were used for PCR analysis of B19R locus. C) Expression of HIV-1 antigens gp120 and GPN. BSC-40 cells were mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At 48 hours post-infection, cells were lysed in Laemmli buffer, cells extracts were fractionated by 10% SDS-PAGE and analyzed by Western blot using rabbit polyclonal anti-gp120 antibody or polyclonal anti-gag p24 serum. D) Replication in CEF cells. CEF cells were infected at an MOI of 0.01 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At different times post-infection (hpi, 0, 24, 48 and 72 hours), cells were harvested, freeze-thawed three times and sonicated. Virus titers in cell lysates were determined by crystal violet staining in BSC-40 cells.
    Bsc 40, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Improved NYVAC-Based Vaccine Vectors"

    Article Title: Improved NYVAC-Based Vaccine Vectors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025674

    A) Scheme of construction. The plasmid transfer vector pGem-RG-B19R wm was obtained by sequential cloning of selectable markers dsRed2 and rsGFP and B19R recombination flanking sequences into the plasmid pGem-7Zf. For the generation of NYVAC-C-ΔB19R, BSC-40 cells were infected at an MOI of 0.01 with NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm. 48 hours post-infection, the cells were harvested, lysed by freeze-thaw cycling, sonicated and used for recombinant virus screening. The deletion mutant was selected from progeny virus by consecutive rounds of plaque purification in BSC-40 cells during which plaques were screened for Red2/GFP fluorescence. B) PCR analysis. Viral DNA was extracted from BSC-40 cells mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. Primers LFB19R-AatII-F and LFB19R-BamHI-R spanning B19R flanking regions were used for PCR analysis of B19R locus. C) Expression of HIV-1 antigens gp120 and GPN. BSC-40 cells were mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At 48 hours post-infection, cells were lysed in Laemmli buffer, cells extracts were fractionated by 10% SDS-PAGE and analyzed by Western blot using rabbit polyclonal anti-gp120 antibody or polyclonal anti-gag p24 serum. D) Replication in CEF cells. CEF cells were infected at an MOI of 0.01 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At different times post-infection (hpi, 0, 24, 48 and 72 hours), cells were harvested, freeze-thawed three times and sonicated. Virus titers in cell lysates were determined by crystal violet staining in BSC-40 cells.
    Figure Legend Snippet: A) Scheme of construction. The plasmid transfer vector pGem-RG-B19R wm was obtained by sequential cloning of selectable markers dsRed2 and rsGFP and B19R recombination flanking sequences into the plasmid pGem-7Zf. For the generation of NYVAC-C-ΔB19R, BSC-40 cells were infected at an MOI of 0.01 with NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm. 48 hours post-infection, the cells were harvested, lysed by freeze-thaw cycling, sonicated and used for recombinant virus screening. The deletion mutant was selected from progeny virus by consecutive rounds of plaque purification in BSC-40 cells during which plaques were screened for Red2/GFP fluorescence. B) PCR analysis. Viral DNA was extracted from BSC-40 cells mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. Primers LFB19R-AatII-F and LFB19R-BamHI-R spanning B19R flanking regions were used for PCR analysis of B19R locus. C) Expression of HIV-1 antigens gp120 and GPN. BSC-40 cells were mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At 48 hours post-infection, cells were lysed in Laemmli buffer, cells extracts were fractionated by 10% SDS-PAGE and analyzed by Western blot using rabbit polyclonal anti-gp120 antibody or polyclonal anti-gag p24 serum. D) Replication in CEF cells. CEF cells were infected at an MOI of 0.01 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At different times post-infection (hpi, 0, 24, 48 and 72 hours), cells were harvested, freeze-thawed three times and sonicated. Virus titers in cell lysates were determined by crystal violet staining in BSC-40 cells.

    Techniques Used: Plasmid Preparation, Clone Assay, Infection, Transfection, Sonication, Recombinant, Mutagenesis, Purification, Fluorescence, Expressing, SDS Page, Western Blot, Staining

    Five groups of mice (n = 6) received an IP inoculation of 2×10 7 PFU/mouse of WR (TK − ), NYVAC-C, NYVAC-C-KC, NYVAC-C-ΔB19R or NYVAC-C-KC-ΔB19R. At different times post-inoculation (24, 48 and 72 hours) animals were sacrificed and different mouse tissues (peritoneal cells, ovaries, livers, spleens and draining lymph nodes) were harvested and processed for titration as described in . The production of infectious virus was determined by plaque assay in BSC-40 cells. The virus titer was expressed as Plaque Forming Units (pfu) per gram of protein.
    Figure Legend Snippet: Five groups of mice (n = 6) received an IP inoculation of 2×10 7 PFU/mouse of WR (TK − ), NYVAC-C, NYVAC-C-KC, NYVAC-C-ΔB19R or NYVAC-C-KC-ΔB19R. At different times post-inoculation (24, 48 and 72 hours) animals were sacrificed and different mouse tissues (peritoneal cells, ovaries, livers, spleens and draining lymph nodes) were harvested and processed for titration as described in . The production of infectious virus was determined by plaque assay in BSC-40 cells. The virus titer was expressed as Plaque Forming Units (pfu) per gram of protein.

    Techniques Used: Titration, Plaque Assay

    epithelial kidney cells  (ATCC)


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    ATCC epithelial kidney cells
    Epithelial Kidney Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bsc 40  (ATCC)


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    ATCC bsc 40
    Bsc 40, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bsc  (ATCC)


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    ATCC bsc
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    dulbecco s modified eagle s medium dmem  (ATCC)


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    ATCC dulbecco s modified eagle s medium dmem
    Dulbecco S Modified Eagle S Medium Dmem, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bsc40 cells  (ATCC)


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    ATCC bsc40 cells
    Recombinant virus containing hTEE-658. ( a ) Schematic outlining the use of homologous recombination to insert the firefly luciferase gene, either with or without hTEE-658 immediately upstream, into the TK locus of the NYVAC strain of VACV. In this strategy, the coding region for GFP is replaced with the inserted sequence of interest to aid in the detection of proper insertion. ( b ) Luciferase expression following infection of <t>BSC40</t> cells with recombinant NYVAC. Luciferase assay results were made relative those when hTEE-658 was not included upstream of the luciferase coding region. Expression experiments were conducted in duplicate, with error reported as standard deviation.
    Bsc40 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Optimization of translation enhancing element use to increase protein expression in a vaccinia virus system"

    Article Title: Optimization of translation enhancing element use to increase protein expression in a vaccinia virus system

    Journal: The Journal of General Virology

    doi: 10.1099/jgv.0.001624

    Recombinant virus containing hTEE-658. ( a ) Schematic outlining the use of homologous recombination to insert the firefly luciferase gene, either with or without hTEE-658 immediately upstream, into the TK locus of the NYVAC strain of VACV. In this strategy, the coding region for GFP is replaced with the inserted sequence of interest to aid in the detection of proper insertion. ( b ) Luciferase expression following infection of BSC40 cells with recombinant NYVAC. Luciferase assay results were made relative those when hTEE-658 was not included upstream of the luciferase coding region. Expression experiments were conducted in duplicate, with error reported as standard deviation.
    Figure Legend Snippet: Recombinant virus containing hTEE-658. ( a ) Schematic outlining the use of homologous recombination to insert the firefly luciferase gene, either with or without hTEE-658 immediately upstream, into the TK locus of the NYVAC strain of VACV. In this strategy, the coding region for GFP is replaced with the inserted sequence of interest to aid in the detection of proper insertion. ( b ) Luciferase expression following infection of BSC40 cells with recombinant NYVAC. Luciferase assay results were made relative those when hTEE-658 was not included upstream of the luciferase coding region. Expression experiments were conducted in duplicate, with error reported as standard deviation.

    Techniques Used: Recombinant, Homologous Recombination, Luciferase, Sequencing, Expressing, Infection, Standard Deviation

    bsc40  (ATCC)


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    ATCC bsc40
    Bsc40, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bsc 40  (ATCC)


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    ATCC bsc 40
    p37-GFP and TIP47 co-immunoprecipitate in the presence of non-ionic detergent . A. Membrane fractions from <t>BSC-40</t> cells infected with vvF13LGFP at an MOI of 5 were extracted as described in Materials and Methods. Protein extracts obtained from the high speed pellet were resuspended in a buffer containing a non-ionic detergent (0.5% NP-40, 150 mM NaCl, 20 mM Tris, pH7.4) and immunoprecipitated with antibodies specific for TIP47 or GFP followed by SDS-PAGE analysis and immunoblot. Antibody raised against the trans Golgi marker, TGN46 (Sigma Aldrich) was used as a non-specific control for immunoprecipitation. Lane 1, IP using TIP47 pab; Lane 2, Unbound Fraction; Lane 3, IP using TGN46 pab; Lane 4, Unbound Fraction; Lane 5, IP using GFP pab, Lane 6, Unbound Fraction, Lane 7, IP using TGN46 pab; Lane 8, Unbound Fraction; B. Immunoblot of whole cell extracts and membrane fractions immunoprecipitated with GFP pab from BSC-40 cells mock-infected or infected with vvWR, vvGFP, vvΔF13LGFP, or vvF13LGFP at an MOI of 5 probed with GFP mab. Lane 1 and 6, mock-infected, Lane 2 and 7, vvWR, Lane 3 and 8, vvF13LGFP, Lane 4 and 9, vvΔF13LGFP, Lane 5 and 10, vvGFP. IP, immunoprecipitated; pab, polyclonal antibody; mab, monoclonal antibody; ** pGFP; * p37-GFP fusion protein; † TIP47 protein.
    Bsc 40, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Vaccinia virus p37 interacts with host proteins associated with LE-derived transport vesicle biogenesis"

    Article Title: Vaccinia virus p37 interacts with host proteins associated with LE-derived transport vesicle biogenesis

    Journal: Virology Journal

    doi: 10.1186/1743-422X-6-44

    p37-GFP and TIP47 co-immunoprecipitate in the presence of non-ionic detergent . A. Membrane fractions from BSC-40 cells infected with vvF13LGFP at an MOI of 5 were extracted as described in Materials and Methods. Protein extracts obtained from the high speed pellet were resuspended in a buffer containing a non-ionic detergent (0.5% NP-40, 150 mM NaCl, 20 mM Tris, pH7.4) and immunoprecipitated with antibodies specific for TIP47 or GFP followed by SDS-PAGE analysis and immunoblot. Antibody raised against the trans Golgi marker, TGN46 (Sigma Aldrich) was used as a non-specific control for immunoprecipitation. Lane 1, IP using TIP47 pab; Lane 2, Unbound Fraction; Lane 3, IP using TGN46 pab; Lane 4, Unbound Fraction; Lane 5, IP using GFP pab, Lane 6, Unbound Fraction, Lane 7, IP using TGN46 pab; Lane 8, Unbound Fraction; B. Immunoblot of whole cell extracts and membrane fractions immunoprecipitated with GFP pab from BSC-40 cells mock-infected or infected with vvWR, vvGFP, vvΔF13LGFP, or vvF13LGFP at an MOI of 5 probed with GFP mab. Lane 1 and 6, mock-infected, Lane 2 and 7, vvWR, Lane 3 and 8, vvF13LGFP, Lane 4 and 9, vvΔF13LGFP, Lane 5 and 10, vvGFP. IP, immunoprecipitated; pab, polyclonal antibody; mab, monoclonal antibody; ** pGFP; * p37-GFP fusion protein; † TIP47 protein.
    Figure Legend Snippet: p37-GFP and TIP47 co-immunoprecipitate in the presence of non-ionic detergent . A. Membrane fractions from BSC-40 cells infected with vvF13LGFP at an MOI of 5 were extracted as described in Materials and Methods. Protein extracts obtained from the high speed pellet were resuspended in a buffer containing a non-ionic detergent (0.5% NP-40, 150 mM NaCl, 20 mM Tris, pH7.4) and immunoprecipitated with antibodies specific for TIP47 or GFP followed by SDS-PAGE analysis and immunoblot. Antibody raised against the trans Golgi marker, TGN46 (Sigma Aldrich) was used as a non-specific control for immunoprecipitation. Lane 1, IP using TIP47 pab; Lane 2, Unbound Fraction; Lane 3, IP using TGN46 pab; Lane 4, Unbound Fraction; Lane 5, IP using GFP pab, Lane 6, Unbound Fraction, Lane 7, IP using TGN46 pab; Lane 8, Unbound Fraction; B. Immunoblot of whole cell extracts and membrane fractions immunoprecipitated with GFP pab from BSC-40 cells mock-infected or infected with vvWR, vvGFP, vvΔF13LGFP, or vvF13LGFP at an MOI of 5 probed with GFP mab. Lane 1 and 6, mock-infected, Lane 2 and 7, vvWR, Lane 3 and 8, vvF13LGFP, Lane 4 and 9, vvΔF13LGFP, Lane 5 and 10, vvGFP. IP, immunoprecipitated; pab, polyclonal antibody; mab, monoclonal antibody; ** pGFP; * p37-GFP fusion protein; † TIP47 protein.

    Techniques Used: Infection, Immunoprecipitation, SDS Page, Western Blot, Marker

    Mutation of the YW motif in F13L blocks association of p37-GFP with TIP47 and prevents complementation of an F13L-deleted virus . (A) Diagram of the VV F13L gene with functional domains indicated. (B) Complementation experiment demonstrating a panel of mutant F13L alleles containing the indicated A or F substitutions. The F13L allelescontained a GFP tag at the C-terminus to facilitate immunoprecipitation of p37. PCR products containing the indicated F13L allele were transfected into BSC-40 cells infected with an F13L-deletion virus. At 3 days post infection/transfection, plaques were visualized by staining with crystal violet, (C) Membrane fractions, prepared as described in Materials and Methods, from infected and transfected cells were immunoprecipitated in hypotonic buffer with anti-GFP antibody and probed with TIP47 antibody (lower panel). The upper panel shows the input controls.
    Figure Legend Snippet: Mutation of the YW motif in F13L blocks association of p37-GFP with TIP47 and prevents complementation of an F13L-deleted virus . (A) Diagram of the VV F13L gene with functional domains indicated. (B) Complementation experiment demonstrating a panel of mutant F13L alleles containing the indicated A or F substitutions. The F13L allelescontained a GFP tag at the C-terminus to facilitate immunoprecipitation of p37. PCR products containing the indicated F13L allele were transfected into BSC-40 cells infected with an F13L-deletion virus. At 3 days post infection/transfection, plaques were visualized by staining with crystal violet, (C) Membrane fractions, prepared as described in Materials and Methods, from infected and transfected cells were immunoprecipitated in hypotonic buffer with anti-GFP antibody and probed with TIP47 antibody (lower panel). The upper panel shows the input controls.

    Techniques Used: Mutagenesis, Functional Assay, Immunoprecipitation, Transfection, Infection, Staining

    ST-246 affects the co-immunoprecipitation of membrane-associated p37-GFP, Rab9 and CI-MPR but not p230 . BSC-40 cells were infected (A and B) with 5 PFU per cell of vvF13LGFP or mock-infected (C) in the absence (column 1) or presence (column 2) of 10 μM ST-246. The membrane fractions were extracted as described in Materials and Methods, resuspended in hypotonic buffer and immunoprecipitated as indicated. Blots were probed with (A) p230 or Rab9 antibody, (B) GFP or Rab9 antibody, and (C) Rab9 antibody. The top row for each are input controls and are probed as indicated.
    Figure Legend Snippet: ST-246 affects the co-immunoprecipitation of membrane-associated p37-GFP, Rab9 and CI-MPR but not p230 . BSC-40 cells were infected (A and B) with 5 PFU per cell of vvF13LGFP or mock-infected (C) in the absence (column 1) or presence (column 2) of 10 μM ST-246. The membrane fractions were extracted as described in Materials and Methods, resuspended in hypotonic buffer and immunoprecipitated as indicated. Blots were probed with (A) p230 or Rab9 antibody, (B) GFP or Rab9 antibody, and (C) Rab9 antibody. The top row for each are input controls and are probed as indicated.

    Techniques Used: Immunoprecipitation, Infection

    ST-246 inhibits membrane precursor biogenesis. BSC-40 cells were infected with 5 PFU per cell of vvF13LGFP in the presence or absence of 10 μM ST-246 . Membrane extracts were prepared as described in Materials and Methods and analyzed by immunoblot (left panel). The remaining supernatant was then subjected to immunoprecipitation (right panel). Blots were probed with the indicated antibodies. Left panel, input controls; right panel, immunoprecipitated samples.
    Figure Legend Snippet: ST-246 inhibits membrane precursor biogenesis. BSC-40 cells were infected with 5 PFU per cell of vvF13LGFP in the presence or absence of 10 μM ST-246 . Membrane extracts were prepared as described in Materials and Methods and analyzed by immunoblot (left panel). The remaining supernatant was then subjected to immunoprecipitation (right panel). Blots were probed with the indicated antibodies. Left panel, input controls; right panel, immunoprecipitated samples.

    Techniques Used: Infection, Western Blot, Immunoprecipitation

    Co-immunoprecipitation and subcellular localization of Rab9 and B5 protein with p37-GFP expressed from vvF13LGFP or an ST-246 resistant VV virus variant . (A) BSC-40 cells were infected with wild type VV (left) or an ST-246-resistant variant that exhibits reduced susceptibility to ST-246 (right) in the presence or absence of 10 μM compound. The membrane fractions were extracted as described in Materials and Methods, resuspended in hypotonic buffer and immunoprecipitated with anti-GFP polyclonal antibody. Blots were probed with Rab9 antibody or antiserum to B5 as indicated. Top row: input controls; (B) Quantitative comparison of immunoblot intensity in the absence and presence of ST-246; (C) Cell monolayers were grown on chamber slides and infected with 8 PFU/cell of an ST-246-resistant variant in the presence or absence of 10 μM ST-246. At 14–16 hpi cells were fixed in 4% paraformaldehyde, permeablized with 0.2% TritonX-100 and stained for 1 hour with anti-p230 antibody or anti-CI-MPR antibody. Proteins were visualized using Alexa 594-conjugated secondary antibody. Samples were mounted in ProLong Gold Antifade Reagent (Invitrogen Molecular Probes) containing DAPI for nuclear staining and analyzed using a Zeiss LSM 510 confocal laser-scanning microscope. Images are representative of experiments in which a minimum of 50 infected cells displaying a similar fluorescent phenotype were observed. Images were collected and processed using LSM 510 acquisition and Adobe Photoshop software. Bar, 5 μm.
    Figure Legend Snippet: Co-immunoprecipitation and subcellular localization of Rab9 and B5 protein with p37-GFP expressed from vvF13LGFP or an ST-246 resistant VV virus variant . (A) BSC-40 cells were infected with wild type VV (left) or an ST-246-resistant variant that exhibits reduced susceptibility to ST-246 (right) in the presence or absence of 10 μM compound. The membrane fractions were extracted as described in Materials and Methods, resuspended in hypotonic buffer and immunoprecipitated with anti-GFP polyclonal antibody. Blots were probed with Rab9 antibody or antiserum to B5 as indicated. Top row: input controls; (B) Quantitative comparison of immunoblot intensity in the absence and presence of ST-246; (C) Cell monolayers were grown on chamber slides and infected with 8 PFU/cell of an ST-246-resistant variant in the presence or absence of 10 μM ST-246. At 14–16 hpi cells were fixed in 4% paraformaldehyde, permeablized with 0.2% TritonX-100 and stained for 1 hour with anti-p230 antibody or anti-CI-MPR antibody. Proteins were visualized using Alexa 594-conjugated secondary antibody. Samples were mounted in ProLong Gold Antifade Reagent (Invitrogen Molecular Probes) containing DAPI for nuclear staining and analyzed using a Zeiss LSM 510 confocal laser-scanning microscope. Images are representative of experiments in which a minimum of 50 infected cells displaying a similar fluorescent phenotype were observed. Images were collected and processed using LSM 510 acquisition and Adobe Photoshop software. Bar, 5 μm.

    Techniques Used: Immunoprecipitation, Variant Assay, Infection, Western Blot, Staining, Laser-Scanning Microscopy, Software

    bsc 40 cells  (ATCC)


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    ATCC bsc 40 cells
    Bsc 40 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bsc40 cells  (ATCC)


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    ATCC bsc40 cells
    A. PI3K inhibitors reduce plaque size in <t>BSC40</t> cells. Cells were infected with 100 PFU of vaccinia virus (VV), strain WR. Inhibitors were added one hour post viral entry. First row: LY294002, 20 µM; B0304, 20 µM; AS1, 50 µM; AS2, 50 µM (LY294002 and B0304 are in 0.2% DMSO and AS1 and AS2 are in 0.5% DMSO); 0.5% DMSO alone. Insets show representative plaques from 50 µM AS1 and 0.5% DMSO treated wells. Second row: 10 µM Wortmannin in 0.1% DMSO did not appear to reduce VV plaque size; 0.1% DMSO control. Results are from three independent trials. B. p85WT and p85α −/− cells form two times more plaques than p85α −/− β −/− cells. Cells were infected with equal amounts of virus and monolayers were stained with crystal violet 48 hours post infection. Results are from eight independent trials. C. PI3K inhibitors do not decrease early viral protein expression. BSC40 cells were infected at pH 7.4 and MOI = 5 with VV and PI3K inhibitors were added 1 hour post-viral entry and allowed to infect for an additional 2 hours. Proteins were subjected to western analysis by anti-E3 mAb and anti-Tubulin mAb. For quantification (right) bands were normalized to tubulin loading control and data is expressed as the fold change relative to DMSO-treated samples. D. Early protein expression is reduced in p85-deficient cells. Cells were infected at an MOI = 5 with VV at pH = 7.4 and analyzed as in C. E. Early protein expression is reduced in p85-deficient cells. Cells were infected at an MOI = 5 with VV. Virus was bound to cells at 4°C, after which cells were moved to 37°C with PBS, pH = 5. Following a 2 hour infection, equal amounts of protein were subjected to western analysis with anti-E3 mAb or anti-Tubulin, and quantitated as in C.
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    1) Product Images from "Multiple Phosphatidylinositol 3-Kinases Regulate Vaccinia Virus Morphogenesis"

    Article Title: Multiple Phosphatidylinositol 3-Kinases Regulate Vaccinia Virus Morphogenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0010884

    A. PI3K inhibitors reduce plaque size in BSC40 cells. Cells were infected with 100 PFU of vaccinia virus (VV), strain WR. Inhibitors were added one hour post viral entry. First row: LY294002, 20 µM; B0304, 20 µM; AS1, 50 µM; AS2, 50 µM (LY294002 and B0304 are in 0.2% DMSO and AS1 and AS2 are in 0.5% DMSO); 0.5% DMSO alone. Insets show representative plaques from 50 µM AS1 and 0.5% DMSO treated wells. Second row: 10 µM Wortmannin in 0.1% DMSO did not appear to reduce VV plaque size; 0.1% DMSO control. Results are from three independent trials. B. p85WT and p85α −/− cells form two times more plaques than p85α −/− β −/− cells. Cells were infected with equal amounts of virus and monolayers were stained with crystal violet 48 hours post infection. Results are from eight independent trials. C. PI3K inhibitors do not decrease early viral protein expression. BSC40 cells were infected at pH 7.4 and MOI = 5 with VV and PI3K inhibitors were added 1 hour post-viral entry and allowed to infect for an additional 2 hours. Proteins were subjected to western analysis by anti-E3 mAb and anti-Tubulin mAb. For quantification (right) bands were normalized to tubulin loading control and data is expressed as the fold change relative to DMSO-treated samples. D. Early protein expression is reduced in p85-deficient cells. Cells were infected at an MOI = 5 with VV at pH = 7.4 and analyzed as in C. E. Early protein expression is reduced in p85-deficient cells. Cells were infected at an MOI = 5 with VV. Virus was bound to cells at 4°C, after which cells were moved to 37°C with PBS, pH = 5. Following a 2 hour infection, equal amounts of protein were subjected to western analysis with anti-E3 mAb or anti-Tubulin, and quantitated as in C.
    Figure Legend Snippet: A. PI3K inhibitors reduce plaque size in BSC40 cells. Cells were infected with 100 PFU of vaccinia virus (VV), strain WR. Inhibitors were added one hour post viral entry. First row: LY294002, 20 µM; B0304, 20 µM; AS1, 50 µM; AS2, 50 µM (LY294002 and B0304 are in 0.2% DMSO and AS1 and AS2 are in 0.5% DMSO); 0.5% DMSO alone. Insets show representative plaques from 50 µM AS1 and 0.5% DMSO treated wells. Second row: 10 µM Wortmannin in 0.1% DMSO did not appear to reduce VV plaque size; 0.1% DMSO control. Results are from three independent trials. B. p85WT and p85α −/− cells form two times more plaques than p85α −/− β −/− cells. Cells were infected with equal amounts of virus and monolayers were stained with crystal violet 48 hours post infection. Results are from eight independent trials. C. PI3K inhibitors do not decrease early viral protein expression. BSC40 cells were infected at pH 7.4 and MOI = 5 with VV and PI3K inhibitors were added 1 hour post-viral entry and allowed to infect for an additional 2 hours. Proteins were subjected to western analysis by anti-E3 mAb and anti-Tubulin mAb. For quantification (right) bands were normalized to tubulin loading control and data is expressed as the fold change relative to DMSO-treated samples. D. Early protein expression is reduced in p85-deficient cells. Cells were infected at an MOI = 5 with VV at pH = 7.4 and analyzed as in C. E. Early protein expression is reduced in p85-deficient cells. Cells were infected at an MOI = 5 with VV. Virus was bound to cells at 4°C, after which cells were moved to 37°C with PBS, pH = 5. Following a 2 hour infection, equal amounts of protein were subjected to western analysis with anti-E3 mAb or anti-Tubulin, and quantitated as in C.

    Techniques Used: Infection, Staining, Expressing, Western Blot

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  • bsc 40  (ATCC)
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    ATCC bsc 40
    A) Scheme of construction. The plasmid transfer vector pGem-RG-B19R wm was obtained by sequential cloning of selectable markers dsRed2 and rsGFP and B19R recombination flanking sequences into the plasmid pGem-7Zf. For the generation of NYVAC-C-ΔB19R, <t>BSC-40</t> cells were infected at an MOI of 0.01 with NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm. 48 hours post-infection, the cells were harvested, lysed by freeze-thaw cycling, sonicated and used for recombinant virus screening. The deletion mutant was selected from progeny virus by consecutive rounds of plaque purification in BSC-40 cells during which plaques were screened for Red2/GFP fluorescence. B) PCR analysis. Viral DNA was extracted from BSC-40 cells mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. Primers LFB19R-AatII-F and LFB19R-BamHI-R spanning B19R flanking regions were used for PCR analysis of B19R locus. C) Expression of HIV-1 antigens gp120 and GPN. BSC-40 cells were mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At 48 hours post-infection, cells were lysed in Laemmli buffer, cells extracts were fractionated by 10% SDS-PAGE and analyzed by Western blot using rabbit polyclonal anti-gp120 antibody or polyclonal anti-gag p24 serum. D) Replication in CEF cells. CEF cells were infected at an MOI of 0.01 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At different times post-infection (hpi, 0, 24, 48 and 72 hours), cells were harvested, freeze-thawed three times and sonicated. Virus titers in cell lysates were determined by crystal violet staining in BSC-40 cells.
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    ATCC epithelial kidney cells
    A) Scheme of construction. The plasmid transfer vector pGem-RG-B19R wm was obtained by sequential cloning of selectable markers dsRed2 and rsGFP and B19R recombination flanking sequences into the plasmid pGem-7Zf. For the generation of NYVAC-C-ΔB19R, <t>BSC-40</t> cells were infected at an MOI of 0.01 with NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm. 48 hours post-infection, the cells were harvested, lysed by freeze-thaw cycling, sonicated and used for recombinant virus screening. The deletion mutant was selected from progeny virus by consecutive rounds of plaque purification in BSC-40 cells during which plaques were screened for Red2/GFP fluorescence. B) PCR analysis. Viral DNA was extracted from BSC-40 cells mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. Primers LFB19R-AatII-F and LFB19R-BamHI-R spanning B19R flanking regions were used for PCR analysis of B19R locus. C) Expression of HIV-1 antigens gp120 and GPN. BSC-40 cells were mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At 48 hours post-infection, cells were lysed in Laemmli buffer, cells extracts were fractionated by 10% SDS-PAGE and analyzed by Western blot using rabbit polyclonal anti-gp120 antibody or polyclonal anti-gag p24 serum. D) Replication in CEF cells. CEF cells were infected at an MOI of 0.01 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At different times post-infection (hpi, 0, 24, 48 and 72 hours), cells were harvested, freeze-thawed three times and sonicated. Virus titers in cell lysates were determined by crystal violet staining in BSC-40 cells.
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    bsc  (ATCC)
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    ATCC bsc
    A) Scheme of construction. The plasmid transfer vector pGem-RG-B19R wm was obtained by sequential cloning of selectable markers dsRed2 and rsGFP and B19R recombination flanking sequences into the plasmid pGem-7Zf. For the generation of NYVAC-C-ΔB19R, <t>BSC-40</t> cells were infected at an MOI of 0.01 with NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm. 48 hours post-infection, the cells were harvested, lysed by freeze-thaw cycling, sonicated and used for recombinant virus screening. The deletion mutant was selected from progeny virus by consecutive rounds of plaque purification in BSC-40 cells during which plaques were screened for Red2/GFP fluorescence. B) PCR analysis. Viral DNA was extracted from BSC-40 cells mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. Primers LFB19R-AatII-F and LFB19R-BamHI-R spanning B19R flanking regions were used for PCR analysis of B19R locus. C) Expression of HIV-1 antigens gp120 and GPN. BSC-40 cells were mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At 48 hours post-infection, cells were lysed in Laemmli buffer, cells extracts were fractionated by 10% SDS-PAGE and analyzed by Western blot using rabbit polyclonal anti-gp120 antibody or polyclonal anti-gag p24 serum. D) Replication in CEF cells. CEF cells were infected at an MOI of 0.01 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At different times post-infection (hpi, 0, 24, 48 and 72 hours), cells were harvested, freeze-thawed three times and sonicated. Virus titers in cell lysates were determined by crystal violet staining in BSC-40 cells.
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    ATCC dulbecco s modified eagle s medium dmem
    A) Scheme of construction. The plasmid transfer vector pGem-RG-B19R wm was obtained by sequential cloning of selectable markers dsRed2 and rsGFP and B19R recombination flanking sequences into the plasmid pGem-7Zf. For the generation of NYVAC-C-ΔB19R, <t>BSC-40</t> cells were infected at an MOI of 0.01 with NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm. 48 hours post-infection, the cells were harvested, lysed by freeze-thaw cycling, sonicated and used for recombinant virus screening. The deletion mutant was selected from progeny virus by consecutive rounds of plaque purification in BSC-40 cells during which plaques were screened for Red2/GFP fluorescence. B) PCR analysis. Viral DNA was extracted from BSC-40 cells mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. Primers LFB19R-AatII-F and LFB19R-BamHI-R spanning B19R flanking regions were used for PCR analysis of B19R locus. C) Expression of HIV-1 antigens gp120 and GPN. BSC-40 cells were mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At 48 hours post-infection, cells were lysed in Laemmli buffer, cells extracts were fractionated by 10% SDS-PAGE and analyzed by Western blot using rabbit polyclonal anti-gp120 antibody or polyclonal anti-gag p24 serum. D) Replication in CEF cells. CEF cells were infected at an MOI of 0.01 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At different times post-infection (hpi, 0, 24, 48 and 72 hours), cells were harvested, freeze-thawed three times and sonicated. Virus titers in cell lysates were determined by crystal violet staining in BSC-40 cells.
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    ATCC bsc40 cells
    Recombinant virus containing hTEE-658. ( a ) Schematic outlining the use of homologous recombination to insert the firefly luciferase gene, either with or without hTEE-658 immediately upstream, into the TK locus of the NYVAC strain of VACV. In this strategy, the coding region for GFP is replaced with the inserted sequence of interest to aid in the detection of proper insertion. ( b ) Luciferase expression following infection of <t>BSC40</t> cells with recombinant NYVAC. Luciferase assay results were made relative those when hTEE-658 was not included upstream of the luciferase coding region. Expression experiments were conducted in duplicate, with error reported as standard deviation.
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    bsc40  (ATCC)
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    ATCC bsc40
    Recombinant virus containing hTEE-658. ( a ) Schematic outlining the use of homologous recombination to insert the firefly luciferase gene, either with or without hTEE-658 immediately upstream, into the TK locus of the NYVAC strain of VACV. In this strategy, the coding region for GFP is replaced with the inserted sequence of interest to aid in the detection of proper insertion. ( b ) Luciferase expression following infection of <t>BSC40</t> cells with recombinant NYVAC. Luciferase assay results were made relative those when hTEE-658 was not included upstream of the luciferase coding region. Expression experiments were conducted in duplicate, with error reported as standard deviation.
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    ATCC bsc 40 cells
    Recombinant virus containing hTEE-658. ( a ) Schematic outlining the use of homologous recombination to insert the firefly luciferase gene, either with or without hTEE-658 immediately upstream, into the TK locus of the NYVAC strain of VACV. In this strategy, the coding region for GFP is replaced with the inserted sequence of interest to aid in the detection of proper insertion. ( b ) Luciferase expression following infection of <t>BSC40</t> cells with recombinant NYVAC. Luciferase assay results were made relative those when hTEE-658 was not included upstream of the luciferase coding region. Expression experiments were conducted in duplicate, with error reported as standard deviation.
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    A) Scheme of construction. The plasmid transfer vector pGem-RG-B19R wm was obtained by sequential cloning of selectable markers dsRed2 and rsGFP and B19R recombination flanking sequences into the plasmid pGem-7Zf. For the generation of NYVAC-C-ΔB19R, BSC-40 cells were infected at an MOI of 0.01 with NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm. 48 hours post-infection, the cells were harvested, lysed by freeze-thaw cycling, sonicated and used for recombinant virus screening. The deletion mutant was selected from progeny virus by consecutive rounds of plaque purification in BSC-40 cells during which plaques were screened for Red2/GFP fluorescence. B) PCR analysis. Viral DNA was extracted from BSC-40 cells mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. Primers LFB19R-AatII-F and LFB19R-BamHI-R spanning B19R flanking regions were used for PCR analysis of B19R locus. C) Expression of HIV-1 antigens gp120 and GPN. BSC-40 cells were mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At 48 hours post-infection, cells were lysed in Laemmli buffer, cells extracts were fractionated by 10% SDS-PAGE and analyzed by Western blot using rabbit polyclonal anti-gp120 antibody or polyclonal anti-gag p24 serum. D) Replication in CEF cells. CEF cells were infected at an MOI of 0.01 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At different times post-infection (hpi, 0, 24, 48 and 72 hours), cells were harvested, freeze-thawed three times and sonicated. Virus titers in cell lysates were determined by crystal violet staining in BSC-40 cells.

    Journal: PLoS ONE

    Article Title: Improved NYVAC-Based Vaccine Vectors

    doi: 10.1371/journal.pone.0025674

    Figure Lengend Snippet: A) Scheme of construction. The plasmid transfer vector pGem-RG-B19R wm was obtained by sequential cloning of selectable markers dsRed2 and rsGFP and B19R recombination flanking sequences into the plasmid pGem-7Zf. For the generation of NYVAC-C-ΔB19R, BSC-40 cells were infected at an MOI of 0.01 with NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm. 48 hours post-infection, the cells were harvested, lysed by freeze-thaw cycling, sonicated and used for recombinant virus screening. The deletion mutant was selected from progeny virus by consecutive rounds of plaque purification in BSC-40 cells during which plaques were screened for Red2/GFP fluorescence. B) PCR analysis. Viral DNA was extracted from BSC-40 cells mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. Primers LFB19R-AatII-F and LFB19R-BamHI-R spanning B19R flanking regions were used for PCR analysis of B19R locus. C) Expression of HIV-1 antigens gp120 and GPN. BSC-40 cells were mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At 48 hours post-infection, cells were lysed in Laemmli buffer, cells extracts were fractionated by 10% SDS-PAGE and analyzed by Western blot using rabbit polyclonal anti-gp120 antibody or polyclonal anti-gag p24 serum. D) Replication in CEF cells. CEF cells were infected at an MOI of 0.01 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At different times post-infection (hpi, 0, 24, 48 and 72 hours), cells were harvested, freeze-thawed three times and sonicated. Virus titers in cell lysates were determined by crystal violet staining in BSC-40 cells.

    Article Snippet: 3×10 6 BSC-40 (ATCC) cells were infected with an MOI of 0.01 of NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm using Lipofectamine (Invitrogen, San Diego, CA).

    Techniques: Plasmid Preparation, Clone Assay, Infection, Transfection, Sonication, Recombinant, Mutagenesis, Purification, Fluorescence, Expressing, SDS Page, Western Blot, Staining

    Five groups of mice (n = 6) received an IP inoculation of 2×10 7 PFU/mouse of WR (TK − ), NYVAC-C, NYVAC-C-KC, NYVAC-C-ΔB19R or NYVAC-C-KC-ΔB19R. At different times post-inoculation (24, 48 and 72 hours) animals were sacrificed and different mouse tissues (peritoneal cells, ovaries, livers, spleens and draining lymph nodes) were harvested and processed for titration as described in . The production of infectious virus was determined by plaque assay in BSC-40 cells. The virus titer was expressed as Plaque Forming Units (pfu) per gram of protein.

    Journal: PLoS ONE

    Article Title: Improved NYVAC-Based Vaccine Vectors

    doi: 10.1371/journal.pone.0025674

    Figure Lengend Snippet: Five groups of mice (n = 6) received an IP inoculation of 2×10 7 PFU/mouse of WR (TK − ), NYVAC-C, NYVAC-C-KC, NYVAC-C-ΔB19R or NYVAC-C-KC-ΔB19R. At different times post-inoculation (24, 48 and 72 hours) animals were sacrificed and different mouse tissues (peritoneal cells, ovaries, livers, spleens and draining lymph nodes) were harvested and processed for titration as described in . The production of infectious virus was determined by plaque assay in BSC-40 cells. The virus titer was expressed as Plaque Forming Units (pfu) per gram of protein.

    Article Snippet: 3×10 6 BSC-40 (ATCC) cells were infected with an MOI of 0.01 of NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm using Lipofectamine (Invitrogen, San Diego, CA).

    Techniques: Titration, Plaque Assay

    Recombinant virus containing hTEE-658. ( a ) Schematic outlining the use of homologous recombination to insert the firefly luciferase gene, either with or without hTEE-658 immediately upstream, into the TK locus of the NYVAC strain of VACV. In this strategy, the coding region for GFP is replaced with the inserted sequence of interest to aid in the detection of proper insertion. ( b ) Luciferase expression following infection of BSC40 cells with recombinant NYVAC. Luciferase assay results were made relative those when hTEE-658 was not included upstream of the luciferase coding region. Expression experiments were conducted in duplicate, with error reported as standard deviation.

    Journal: The Journal of General Virology

    Article Title: Optimization of translation enhancing element use to increase protein expression in a vaccinia virus system

    doi: 10.1099/jgv.0.001624

    Figure Lengend Snippet: Recombinant virus containing hTEE-658. ( a ) Schematic outlining the use of homologous recombination to insert the firefly luciferase gene, either with or without hTEE-658 immediately upstream, into the TK locus of the NYVAC strain of VACV. In this strategy, the coding region for GFP is replaced with the inserted sequence of interest to aid in the detection of proper insertion. ( b ) Luciferase expression following infection of BSC40 cells with recombinant NYVAC. Luciferase assay results were made relative those when hTEE-658 was not included upstream of the luciferase coding region. Expression experiments were conducted in duplicate, with error reported as standard deviation.

    Article Snippet: HeLa and BSC40 cells were obtained from American Type Culture Collection (ATCC).

    Techniques: Recombinant, Homologous Recombination, Luciferase, Sequencing, Expressing, Infection, Standard Deviation