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  • 92
    Lonza optimem
    Optimem, supplied by Lonza, used in various techniques. Bioz Stars score: 92/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/optimem/product/Lonza
    Average 92 stars, based on 76 article reviews
    Price from $9.99 to $1999.99
    optimem - by Bioz Stars, 2020-08
    92/100 stars
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    93
    Millipore optimem
    Optimem, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 518 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/optimem/product/Millipore
    Average 93 stars, based on 518 article reviews
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    optimem - by Bioz Stars, 2020-08
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    94
    Thermo Fisher optimem
    Actin is required for ABLV G-mediated viral entry. (A) HEK293T cell monolayers were pretreated with latrunculin B (LatB) diluted in <t>OptiMEM®</t> for 1 hr at 37°C. Cells were then infected with max-GFP encoding rVSV reporter viruses (MOI = 3). Under these conditions, a MOI of 3 yielded at least 50% virus-infected cells in untreated controls. Cells were harvested 8 hrs post infection and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. Reporter viruses that express VSV G were included as a positive control to assess LatB activity. Results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are standard error of the mean (SEM).
    Optimem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 21420 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/optimem/product/Thermo Fisher
    Average 94 stars, based on 21420 article reviews
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    optimem - by Bioz Stars, 2020-08
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    92
    GE Healthcare optimem
    Actin is required for ABLV G-mediated viral entry. (A) HEK293T cell monolayers were pretreated with latrunculin B (LatB) diluted in <t>OptiMEM®</t> for 1 hr at 37°C. Cells were then infected with max-GFP encoding rVSV reporter viruses (MOI = 3). Under these conditions, a MOI of 3 yielded at least 50% virus-infected cells in untreated controls. Cells were harvested 8 hrs post infection and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. Reporter viruses that express VSV G were included as a positive control to assess LatB activity. Results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are standard error of the mean (SEM).
    Optimem, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/optimem/product/GE Healthcare
    Average 92 stars, based on 46 article reviews
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    optimem - by Bioz Stars, 2020-08
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    92
    promega optimem
    Actin is required for ABLV G-mediated viral entry. (A) HEK293T cell monolayers were pretreated with latrunculin B (LatB) diluted in <t>OptiMEM®</t> for 1 hr at 37°C. Cells were then infected with max-GFP encoding rVSV reporter viruses (MOI = 3). Under these conditions, a MOI of 3 yielded at least 50% virus-infected cells in untreated controls. Cells were harvested 8 hrs post infection and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. Reporter viruses that express VSV G were included as a positive control to assess LatB activity. Results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are standard error of the mean (SEM).
    Optimem, supplied by promega, used in various techniques. Bioz Stars score: 92/100, based on 169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 169 article reviews
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    optimem - by Bioz Stars, 2020-08
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    92
    Corning Life Sciences optimem
    Actin is required for ABLV G-mediated viral entry. (A) HEK293T cell monolayers were pretreated with latrunculin B (LatB) diluted in <t>OptiMEM®</t> for 1 hr at 37°C. Cells were then infected with max-GFP encoding rVSV reporter viruses (MOI = 3). Under these conditions, a MOI of 3 yielded at least 50% virus-infected cells in untreated controls. Cells were harvested 8 hrs post infection and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. Reporter viruses that express VSV G were included as a positive control to assess LatB activity. Results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are standard error of the mean (SEM).
    Optimem, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 39 article reviews
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    optimem - by Bioz Stars, 2020-08
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    92
    PAA Laboratories optimem
    Actin is required for ABLV G-mediated viral entry. (A) HEK293T cell monolayers were pretreated with latrunculin B (LatB) diluted in <t>OptiMEM®</t> for 1 hr at 37°C. Cells were then infected with max-GFP encoding rVSV reporter viruses (MOI = 3). Under these conditions, a MOI of 3 yielded at least 50% virus-infected cells in untreated controls. Cells were harvested 8 hrs post infection and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. Reporter viruses that express VSV G were included as a positive control to assess LatB activity. Results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are standard error of the mean (SEM).
    Optimem, supplied by PAA Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 46 article reviews
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    optimem - by Bioz Stars, 2020-08
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    92
    Fisher Scientific optimem
    Apparent ferroxidase activity derived from culture medium. Media of interest was tested by the triplex assay to measure loss of Fe 2+ ( A ), conversion of Fe 3+ ( B ) and loading of Fe 3+ into TF ( C ). Without apo-TF, a one in five dilution of <t>OptiMEM,</t> neurobasal (NB) and RPMI-1640 (RPMI) media oxidized Fe 2+ to Fe 3+ ( A B ). The addition of apo-TF (50 µM) promoted Fe 2+ oxidation by all media ( A ), and had comparable ability to load Fe 3+ onto TF ( C ). Assays were run at 26°C in HBS, pH = 7.2+ FAS (100 µM) ± apo-TF (50 µM). Individual data points were mean ± S.E. of 2 experiments, performed in duplicate.
    Optimem, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/optimem/product/Fisher Scientific
    Average 92 stars, based on 28 article reviews
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    optimem - by Bioz Stars, 2020-08
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    85
    Thermo Fisher 50ul optimem
    Apparent ferroxidase activity derived from culture medium. Media of interest was tested by the triplex assay to measure loss of Fe 2+ ( A ), conversion of Fe 3+ ( B ) and loading of Fe 3+ into TF ( C ). Without apo-TF, a one in five dilution of <t>OptiMEM,</t> neurobasal (NB) and RPMI-1640 (RPMI) media oxidized Fe 2+ to Fe 3+ ( A B ). The addition of apo-TF (50 µM) promoted Fe 2+ oxidation by all media ( A ), and had comparable ability to load Fe 3+ onto TF ( C ). Assays were run at 26°C in HBS, pH = 7.2+ FAS (100 µM) ± apo-TF (50 µM). Individual data points were mean ± S.E. of 2 experiments, performed in duplicate.
    50ul Optimem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    50ul optimem - by Bioz Stars, 2020-08
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    89
    Thermo Fisher optimem glutamax
    Apparent ferroxidase activity derived from culture medium. Media of interest was tested by the triplex assay to measure loss of Fe 2+ ( A ), conversion of Fe 3+ ( B ) and loading of Fe 3+ into TF ( C ). Without apo-TF, a one in five dilution of <t>OptiMEM,</t> neurobasal (NB) and RPMI-1640 (RPMI) media oxidized Fe 2+ to Fe 3+ ( A B ). The addition of apo-TF (50 µM) promoted Fe 2+ oxidation by all media ( A ), and had comparable ability to load Fe 3+ onto TF ( C ). Assays were run at 26°C in HBS, pH = 7.2+ FAS (100 µM) ± apo-TF (50 µM). Individual data points were mean ± S.E. of 2 experiments, performed in duplicate.
    Optimem Glutamax, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher optimem glutimax
    Apparent ferroxidase activity derived from culture medium. Media of interest was tested by the triplex assay to measure loss of Fe 2+ ( A ), conversion of Fe 3+ ( B ) and loading of Fe 3+ into TF ( C ). Without apo-TF, a one in five dilution of <t>OptiMEM,</t> neurobasal (NB) and RPMI-1640 (RPMI) media oxidized Fe 2+ to Fe 3+ ( A B ). The addition of apo-TF (50 µM) promoted Fe 2+ oxidation by all media ( A ), and had comparable ability to load Fe 3+ onto TF ( C ). Assays were run at 26°C in HBS, pH = 7.2+ FAS (100 µM) ± apo-TF (50 µM). Individual data points were mean ± S.E. of 2 experiments, performed in duplicate.
    Optimem Glutimax, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    optimem glutimax - by Bioz Stars, 2020-08
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    92
    Thermo Fisher plain optimem
    Apparent ferroxidase activity derived from culture medium. Media of interest was tested by the triplex assay to measure loss of Fe 2+ ( A ), conversion of Fe 3+ ( B ) and loading of Fe 3+ into TF ( C ). Without apo-TF, a one in five dilution of <t>OptiMEM,</t> neurobasal (NB) and RPMI-1640 (RPMI) media oxidized Fe 2+ to Fe 3+ ( A B ). The addition of apo-TF (50 µM) promoted Fe 2+ oxidation by all media ( A ), and had comparable ability to load Fe 3+ onto TF ( C ). Assays were run at 26°C in HBS, pH = 7.2+ FAS (100 µM) ± apo-TF (50 µM). Individual data points were mean ± S.E. of 2 experiments, performed in duplicate.
    Plain Optimem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher 200ul optimem
    Apparent ferroxidase activity derived from culture medium. Media of interest was tested by the triplex assay to measure loss of Fe 2+ ( A ), conversion of Fe 3+ ( B ) and loading of Fe 3+ into TF ( C ). Without apo-TF, a one in five dilution of <t>OptiMEM,</t> neurobasal (NB) and RPMI-1640 (RPMI) media oxidized Fe 2+ to Fe 3+ ( A B ). The addition of apo-TF (50 µM) promoted Fe 2+ oxidation by all media ( A ), and had comparable ability to load Fe 3+ onto TF ( C ). Assays were run at 26°C in HBS, pH = 7.2+ FAS (100 µM) ± apo-TF (50 µM). Individual data points were mean ± S.E. of 2 experiments, performed in duplicate.
    200ul Optimem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    200ul optimem - by Bioz Stars, 2020-08
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    85
    Thermo Fisher iced optimem
    Apparent ferroxidase activity derived from culture medium. Media of interest was tested by the triplex assay to measure loss of Fe 2+ ( A ), conversion of Fe 3+ ( B ) and loading of Fe 3+ into TF ( C ). Without apo-TF, a one in five dilution of <t>OptiMEM,</t> neurobasal (NB) and RPMI-1640 (RPMI) media oxidized Fe 2+ to Fe 3+ ( A B ). The addition of apo-TF (50 µM) promoted Fe 2+ oxidation by all media ( A ), and had comparable ability to load Fe 3+ onto TF ( C ). Assays were run at 26°C in HBS, pH = 7.2+ FAS (100 µM) ± apo-TF (50 µM). Individual data points were mean ± S.E. of 2 experiments, performed in duplicate.
    Iced Optimem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher optimem f
    Apparent ferroxidase activity derived from culture medium. Media of interest was tested by the triplex assay to measure loss of Fe 2+ ( A ), conversion of Fe 3+ ( B ) and loading of Fe 3+ into TF ( C ). Without apo-TF, a one in five dilution of <t>OptiMEM,</t> neurobasal (NB) and RPMI-1640 (RPMI) media oxidized Fe 2+ to Fe 3+ ( A B ). The addition of apo-TF (50 µM) promoted Fe 2+ oxidation by all media ( A ), and had comparable ability to load Fe 3+ onto TF ( C ). Assays were run at 26°C in HBS, pH = 7.2+ FAS (100 µM) ± apo-TF (50 µM). Individual data points were mean ± S.E. of 2 experiments, performed in duplicate.
    Optimem F, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher 1×optimem
    Apparent ferroxidase activity derived from culture medium. Media of interest was tested by the triplex assay to measure loss of Fe 2+ ( A ), conversion of Fe 3+ ( B ) and loading of Fe 3+ into TF ( C ). Without apo-TF, a one in five dilution of <t>OptiMEM,</t> neurobasal (NB) and RPMI-1640 (RPMI) media oxidized Fe 2+ to Fe 3+ ( A B ). The addition of apo-TF (50 µM) promoted Fe 2+ oxidation by all media ( A ), and had comparable ability to load Fe 3+ onto TF ( C ). Assays were run at 26°C in HBS, pH = 7.2+ FAS (100 µM) ± apo-TF (50 µM). Individual data points were mean ± S.E. of 2 experiments, performed in duplicate.
    1×Optimem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher optimem buffer
    Apparent ferroxidase activity derived from culture medium. Media of interest was tested by the triplex assay to measure loss of Fe 2+ ( A ), conversion of Fe 3+ ( B ) and loading of Fe 3+ into TF ( C ). Without apo-TF, a one in five dilution of <t>OptiMEM,</t> neurobasal (NB) and RPMI-1640 (RPMI) media oxidized Fe 2+ to Fe 3+ ( A B ). The addition of apo-TF (50 µM) promoted Fe 2+ oxidation by all media ( A ), and had comparable ability to load Fe 3+ onto TF ( C ). Assays were run at 26°C in HBS, pH = 7.2+ FAS (100 µM) ± apo-TF (50 µM). Individual data points were mean ± S.E. of 2 experiments, performed in duplicate.
    Optimem Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher sterile optimem
    Apparent ferroxidase activity derived from culture medium. Media of interest was tested by the triplex assay to measure loss of Fe 2+ ( A ), conversion of Fe 3+ ( B ) and loading of Fe 3+ into TF ( C ). Without apo-TF, a one in five dilution of <t>OptiMEM,</t> neurobasal (NB) and RPMI-1640 (RPMI) media oxidized Fe 2+ to Fe 3+ ( A B ). The addition of apo-TF (50 µM) promoted Fe 2+ oxidation by all media ( A ), and had comparable ability to load Fe 3+ onto TF ( C ). Assays were run at 26°C in HBS, pH = 7.2+ FAS (100 µM) ± apo-TF (50 µM). Individual data points were mean ± S.E. of 2 experiments, performed in duplicate.
    Sterile Optimem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Actin is required for ABLV G-mediated viral entry. (A) HEK293T cell monolayers were pretreated with latrunculin B (LatB) diluted in OptiMEM® for 1 hr at 37°C. Cells were then infected with max-GFP encoding rVSV reporter viruses (MOI = 3). Under these conditions, a MOI of 3 yielded at least 50% virus-infected cells in untreated controls. Cells were harvested 8 hrs post infection and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. Reporter viruses that express VSV G were included as a positive control to assess LatB activity. Results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are standard error of the mean (SEM).

    Journal: Virology Journal

    Article Title: Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5

    doi: 10.1186/1743-422X-11-40

    Figure Lengend Snippet: Actin is required for ABLV G-mediated viral entry. (A) HEK293T cell monolayers were pretreated with latrunculin B (LatB) diluted in OptiMEM® for 1 hr at 37°C. Cells were then infected with max-GFP encoding rVSV reporter viruses (MOI = 3). Under these conditions, a MOI of 3 yielded at least 50% virus-infected cells in untreated controls. Cells were harvested 8 hrs post infection and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. Reporter viruses that express VSV G were included as a positive control to assess LatB activity. Results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are standard error of the mean (SEM).

    Article Snippet: All drugs and viruses were diluted in OptiMEM® (Invitrogen, Carlsbad, CA) and drugs were maintained for the entire course of infection.

    Techniques: Infection, Staining, Positive Control, Activity Assay

    Chemical inhibition of CME inhibits ABLV G-mediated viral entry into HEK293T cells. (A) HEK293T monolayers were pretreated with chlorpromazine diluted in OptiMEM® for 30 min at 37°C. Cells were then infected with rVSV (MOI = 3) for 8 hrs and then analyzed as described in Figure 1 . Under these conditions, a MOI of 3 yielded 50-60% virus-infected cells in untreated cells. Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. The rVSV-VSV G reporter virus was included as positive control. Results are expressed as percent virus-infected cells relative to that of controls and represent 3 independent experiments; error bars are SEM.

    Journal: Virology Journal

    Article Title: Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5

    doi: 10.1186/1743-422X-11-40

    Figure Lengend Snippet: Chemical inhibition of CME inhibits ABLV G-mediated viral entry into HEK293T cells. (A) HEK293T monolayers were pretreated with chlorpromazine diluted in OptiMEM® for 30 min at 37°C. Cells were then infected with rVSV (MOI = 3) for 8 hrs and then analyzed as described in Figure 1 . Under these conditions, a MOI of 3 yielded 50-60% virus-infected cells in untreated cells. Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. The rVSV-VSV G reporter virus was included as positive control. Results are expressed as percent virus-infected cells relative to that of controls and represent 3 independent experiments; error bars are SEM.

    Article Snippet: All drugs and viruses were diluted in OptiMEM® (Invitrogen, Carlsbad, CA) and drugs were maintained for the entire course of infection.

    Techniques: Inhibition, Infection, Staining, Positive Control

    CavME endocytosis and macropinocytosis are not required for ABLV G-mediated viral entry. (A) Chemical inhibition of CavME. HEK293T cell monolayers were pretreated with filipin diluted in OptiMEM® for 1 hr at 37°C. Cells were then infected with rVSV (MOI = 1) for 20 hrs and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection. (B) Cholera toxin B (CTX-B) subunit uptake is inhibited by filipin. Following pretreatment with filipin as described in (A) , HEK293T cell monolayers grown on 12 mm coverslips were incubated with Alexa Fluor 488-labeled CTX-B (10 μg/ml) for 1 hr at 37°C. Cells were then washed twice with PBS, fixed and imaged. Images were taken by confocal microscopy with a mid z-section shown. Nuclei were stained with DAPI, (4′,6-diamidino-2-phenylindole, dihydrochloride). (C) Chemical inhibition of macropinocytosis. HEK293T cell monolayers were pretreated with EIPA diluted in OptiMEM® for 1 hr at 37°C. Cells were then infected with rVSV (MOI = 1) for 20 hrs and analyzed as described in Figure 1 . rVSV encoding VSV G (MOI = 1) or EboGP (MOI = 15) were included as negative and positive controls, respectively, to assess EIPA activity. Drug was maintained for the entire course of infection. For (A) and (C) results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are SEM. Under these experimental conditions, the chosen MOIs yielded 60-70% virus-infected cells in untreated controls. EIPA, 5-(N-ethyl-N-isopropyl) amiloride.

    Journal: Virology Journal

    Article Title: Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5

    doi: 10.1186/1743-422X-11-40

    Figure Lengend Snippet: CavME endocytosis and macropinocytosis are not required for ABLV G-mediated viral entry. (A) Chemical inhibition of CavME. HEK293T cell monolayers were pretreated with filipin diluted in OptiMEM® for 1 hr at 37°C. Cells were then infected with rVSV (MOI = 1) for 20 hrs and analyzed as described in Figure 1 . Drug was maintained for the entire course of infection. (B) Cholera toxin B (CTX-B) subunit uptake is inhibited by filipin. Following pretreatment with filipin as described in (A) , HEK293T cell monolayers grown on 12 mm coverslips were incubated with Alexa Fluor 488-labeled CTX-B (10 μg/ml) for 1 hr at 37°C. Cells were then washed twice with PBS, fixed and imaged. Images were taken by confocal microscopy with a mid z-section shown. Nuclei were stained with DAPI, (4′,6-diamidino-2-phenylindole, dihydrochloride). (C) Chemical inhibition of macropinocytosis. HEK293T cell monolayers were pretreated with EIPA diluted in OptiMEM® for 1 hr at 37°C. Cells were then infected with rVSV (MOI = 1) for 20 hrs and analyzed as described in Figure 1 . rVSV encoding VSV G (MOI = 1) or EboGP (MOI = 15) were included as negative and positive controls, respectively, to assess EIPA activity. Drug was maintained for the entire course of infection. For (A) and (C) results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are SEM. Under these experimental conditions, the chosen MOIs yielded 60-70% virus-infected cells in untreated controls. EIPA, 5-(N-ethyl-N-isopropyl) amiloride.

    Article Snippet: All drugs and viruses were diluted in OptiMEM® (Invitrogen, Carlsbad, CA) and drugs were maintained for the entire course of infection.

    Techniques: Inhibition, Infection, Incubation, Labeling, Confocal Microscopy, Staining, Activity Assay

    Chemical inhibition of dynamin inhibits ABLV G-mediated viral entry into HEK293T cells. (A) HEK293T cell monolayers were pretreated with dynasore diluted in OptiMEM® for 30 min at 37°C and were then infected with max-GFP encoding rVSV reporter viruses (MOI = 1). Cells were harvested 20 hrs post infection, fixed with 2% paraformaldehyde and then analyzed for GFP expression (indicative of productive infection). GFP positive cells were counted with a Nexcelom Vision automated cell counter with fluorescence detection and the percent of virus-infected cells was calculated by dividing the number of GFP positive cells by the total number of cells counted. Under these experimental conditions, a MOI of 1 yielded 60-70% virus-infected cells in untreated cells. Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. Reporter viruses that express VSV G were included as a positive control to assess dynasore activity. Results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are standard error of the mean (SEM).

    Journal: Virology Journal

    Article Title: Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5

    doi: 10.1186/1743-422X-11-40

    Figure Lengend Snippet: Chemical inhibition of dynamin inhibits ABLV G-mediated viral entry into HEK293T cells. (A) HEK293T cell monolayers were pretreated with dynasore diluted in OptiMEM® for 30 min at 37°C and were then infected with max-GFP encoding rVSV reporter viruses (MOI = 1). Cells were harvested 20 hrs post infection, fixed with 2% paraformaldehyde and then analyzed for GFP expression (indicative of productive infection). GFP positive cells were counted with a Nexcelom Vision automated cell counter with fluorescence detection and the percent of virus-infected cells was calculated by dividing the number of GFP positive cells by the total number of cells counted. Under these experimental conditions, a MOI of 1 yielded 60-70% virus-infected cells in untreated cells. Drug was maintained for the entire course of infection and its effect on cell viability (B) was determined by trypan blue staining. Reporter viruses that express VSV G were included as a positive control to assess dynasore activity. Results are expressed as percent virus-infected cells relative to that of untreated controls and represent 3 independent experiments; error bars are standard error of the mean (SEM).

    Article Snippet: All drugs and viruses were diluted in OptiMEM® (Invitrogen, Carlsbad, CA) and drugs were maintained for the entire course of infection.

    Techniques: Inhibition, Infection, Expressing, Fluorescence, Staining, Positive Control, Activity Assay

    Generation of a human corneal endothelium cell (HCEC) vehicle for cell therapy. (A) Effect of various media types on HCEC adhesion to the substrate. HCECs were seeded with media without supplementation with FBS, and then the numbers of adhering cell were evaluated after 24 hours. RELAR, M199, and F12/DMEM media significantly enhanced cell adhesion when compared to OptiMEM-I. * p

    Journal: PLoS ONE

    Article Title: Generation and Feasibility Assessment of a New Vehicle for Cell-Based Therapy for Treating Corneal Endothelial Dysfunction

    doi: 10.1371/journal.pone.0158427

    Figure Lengend Snippet: Generation of a human corneal endothelium cell (HCEC) vehicle for cell therapy. (A) Effect of various media types on HCEC adhesion to the substrate. HCECs were seeded with media without supplementation with FBS, and then the numbers of adhering cell were evaluated after 24 hours. RELAR, M199, and F12/DMEM media significantly enhanced cell adhesion when compared to OptiMEM-I. * p

    Article Snippet: First, basal medium was prepared, consisting of OptiMEM-I (Life Technologies Corp., Carlsbad, CA), 8% FBS, 5 ng/mL epidermal growth factor (Sigma-Aldrich Co., St. Louis, MO), 20 μg/mL ascorbic acid (Sigma-Aldrich Co.), 200 mg/L calcium chloride, 0.08% chondroitin sulfate (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 50 μg/mL gentamicin, and 10μM SB431542 (Merck Millipore, Billerica, MA).

    Techniques:

    Apparent ferroxidase activity derived from culture medium. Media of interest was tested by the triplex assay to measure loss of Fe 2+ ( A ), conversion of Fe 3+ ( B ) and loading of Fe 3+ into TF ( C ). Without apo-TF, a one in five dilution of OptiMEM, neurobasal (NB) and RPMI-1640 (RPMI) media oxidized Fe 2+ to Fe 3+ ( A B ). The addition of apo-TF (50 µM) promoted Fe 2+ oxidation by all media ( A ), and had comparable ability to load Fe 3+ onto TF ( C ). Assays were run at 26°C in HBS, pH = 7.2+ FAS (100 µM) ± apo-TF (50 µM). Individual data points were mean ± S.E. of 2 experiments, performed in duplicate.

    Journal: PLoS ONE

    Article Title: β-Amyloid Precursor Protein Does Not Possess Ferroxidase Activity but Does Stabilize the Cell Surface Ferrous Iron Exporter Ferroportin

    doi: 10.1371/journal.pone.0114174

    Figure Lengend Snippet: Apparent ferroxidase activity derived from culture medium. Media of interest was tested by the triplex assay to measure loss of Fe 2+ ( A ), conversion of Fe 3+ ( B ) and loading of Fe 3+ into TF ( C ). Without apo-TF, a one in five dilution of OptiMEM, neurobasal (NB) and RPMI-1640 (RPMI) media oxidized Fe 2+ to Fe 3+ ( A B ). The addition of apo-TF (50 µM) promoted Fe 2+ oxidation by all media ( A ), and had comparable ability to load Fe 3+ onto TF ( C ). Assays were run at 26°C in HBS, pH = 7.2+ FAS (100 µM) ± apo-TF (50 µM). Individual data points were mean ± S.E. of 2 experiments, performed in duplicate.

    Article Snippet: In turn, this was added to 1 mL of OptiMEM containing 20 µL of Dharmafect 1 (Fisher Scientific Ltd), prepared separately.

    Techniques: Activity Assay, Derivative Assay

    Extracellular sAPPα or CP increases cellular expression of surface ferroportin. FPN location was examined in ( A ) HEK293T and ( B - D ) primary murine neuronal cultures, preincubated with iron (50 µM, 3 h) followed with CP or sAPPα (1 µM, 30 min). Both cell lines have been previously shown to utilize APP to promote iron efflux, and do not express CP [2] , [14] . Surface proteins on ( A ) HEK293T cells, and ( B ) primary neurons, were biotinylated to identify changes to endogenous FPN and APP expression on the cell surface, as well as exogenously attached sAPPα or CP. Surface levels of FPN were significantly increased in the presence of CP or sAPPα, despite total levels of FPN remaining unchanged. The graphs show the distribution of FPN when normalized against the β-actin content of the intracellular+surface fractions, and adjusted for protein load. Similar results for FPN distribution were achieved even without adjusting for β-actin (not shown). ( C ) Fluorescence-activated cell sorting of non-permeabilized N2a neuroblastoma cultures preincubated with iron (50 µM, 6 h) confirms an increase in surface expression of FPN, quantified in ( D ), after a 30 min incubation with sAPPα (1 µM) in OptiMEM. ( E ) Deconvoluted confocal microscopy shows overlap of endogenous APP and FPN at the surface of non-permeabilized primary neurons preincubated with iron (50 µM, 3 h), as well as ( F ) increased FPN on the neuronal surface following further treatment with CP or sAPPα (1 µM, 30 min). Endogenous surface FPN was below detection limits in neurons that were not treated with FAS (not shown). Data in ( A ), ( B ) ( D ) are means ± S.E. of 3 experiments, performed in duplicate. * p

    Journal: PLoS ONE

    Article Title: β-Amyloid Precursor Protein Does Not Possess Ferroxidase Activity but Does Stabilize the Cell Surface Ferrous Iron Exporter Ferroportin

    doi: 10.1371/journal.pone.0114174

    Figure Lengend Snippet: Extracellular sAPPα or CP increases cellular expression of surface ferroportin. FPN location was examined in ( A ) HEK293T and ( B - D ) primary murine neuronal cultures, preincubated with iron (50 µM, 3 h) followed with CP or sAPPα (1 µM, 30 min). Both cell lines have been previously shown to utilize APP to promote iron efflux, and do not express CP [2] , [14] . Surface proteins on ( A ) HEK293T cells, and ( B ) primary neurons, were biotinylated to identify changes to endogenous FPN and APP expression on the cell surface, as well as exogenously attached sAPPα or CP. Surface levels of FPN were significantly increased in the presence of CP or sAPPα, despite total levels of FPN remaining unchanged. The graphs show the distribution of FPN when normalized against the β-actin content of the intracellular+surface fractions, and adjusted for protein load. Similar results for FPN distribution were achieved even without adjusting for β-actin (not shown). ( C ) Fluorescence-activated cell sorting of non-permeabilized N2a neuroblastoma cultures preincubated with iron (50 µM, 6 h) confirms an increase in surface expression of FPN, quantified in ( D ), after a 30 min incubation with sAPPα (1 µM) in OptiMEM. ( E ) Deconvoluted confocal microscopy shows overlap of endogenous APP and FPN at the surface of non-permeabilized primary neurons preincubated with iron (50 µM, 3 h), as well as ( F ) increased FPN on the neuronal surface following further treatment with CP or sAPPα (1 µM, 30 min). Endogenous surface FPN was below detection limits in neurons that were not treated with FAS (not shown). Data in ( A ), ( B ) ( D ) are means ± S.E. of 3 experiments, performed in duplicate. * p

    Article Snippet: In turn, this was added to 1 mL of OptiMEM containing 20 µL of Dharmafect 1 (Fisher Scientific Ltd), prepared separately.

    Techniques: Expressing, Fluorescence, FACS, Incubation, Confocal Microscopy