goat anti mouse  (Thermo Fisher)


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    Name:
    Goat anti Mouse IgG
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    Catalog Number:
    PA1-32125
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    Structured Review

    Thermo Fisher goat anti mouse

    https://www.bioz.com/result/goat anti mouse/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti mouse - by Bioz Stars, 2021-04
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    Expressing:

    Article Title: Systemic treatment with CAR-engineered T cells against PSCA delays subcutaneous tumor growth and prolongs survival of mice
    Article Snippet: Activated cells were transduced with 50 μl concentrated PSCA-CAR-encoding lentivirus or Mock lentivirus for 4 hours at 37°C in the presence of 10 μg/ml protamine sulphate and 100 IU IL-2 (Sigma-Aldrich). .. For analysis of PSCA-CAR expression, cells were stained with biotinylated protein-L (Genscript, Piscataway, NJ) [ ], washed 3 times with PBS containing 4% BSA, followed by labeling with phycoerythrin (PE)-conjugated streptavidin (BD Biosciences) or stained with Alexa Fluor® 647 F(ab')2 Fragment of Goat Anti-Mouse IgG (H + L) (Invitrogen) and stained with an allophycocyanin (APC)-conjugated anti-CD3 or fluorescein isothiocyanate (FITC)-conjugated anti-CD3 antibody (Nordic Biosite). .. Flow cytometry analysis was performed using FACSCanto II or LSRII (BD Biosciences).

    Staining:

    Article Title: Systemic treatment with CAR-engineered T cells against PSCA delays subcutaneous tumor growth and prolongs survival of mice
    Article Snippet: Activated cells were transduced with 50 μl concentrated PSCA-CAR-encoding lentivirus or Mock lentivirus for 4 hours at 37°C in the presence of 10 μg/ml protamine sulphate and 100 IU IL-2 (Sigma-Aldrich). .. For analysis of PSCA-CAR expression, cells were stained with biotinylated protein-L (Genscript, Piscataway, NJ) [ ], washed 3 times with PBS containing 4% BSA, followed by labeling with phycoerythrin (PE)-conjugated streptavidin (BD Biosciences) or stained with Alexa Fluor® 647 F(ab')2 Fragment of Goat Anti-Mouse IgG (H + L) (Invitrogen) and stained with an allophycocyanin (APC)-conjugated anti-CD3 or fluorescein isothiocyanate (FITC)-conjugated anti-CD3 antibody (Nordic Biosite). .. Flow cytometry analysis was performed using FACSCanto II or LSRII (BD Biosciences).

    Article Title: Anti-C1q autoantibodies deposit in glomeruli but are only pathogenic in combination with glomerular C1q-containing immune complexes
    Article Snippet: .. Sections were stained for the presence of mouse IgG, rabbit IgG, and mouse C1q using goat anti-mouse IgG Oregon Green (Invitrogen Corp.), goat anti-rabbit IgG conjugated to FITC (Nordic Immunological Laboratories), and rabbit anti–mouse C1q DIG, followed by sheep anti-DIG conjugated to HRP, where appropriate. .. For double staining, goat anti-mouse IgG Alexa 546 (Invitrogen Corp.) was used in combination with anti-C1q and anti-rabbit IgG stainings, as described above.

    Article Title: Identification of the Mycobacterium ulcerans Protein MUL_3720 as a Promising Target for the Development of a Diagnostic Test for Buruli Ulcer
    Article Snippet: Agarose blocks were embedded into paraffin, cut in 3 μm sections and transferred onto microscopy glass slides (Thermo Scientific). .. Bacteria were stained with mAb JD3.2 and Alexa fluor488 (Invitrogen) conjugated goat anti-mouse IgG and mounted in ProLong Gold anti-fade reagent containing 4′,6-Diamidino-2-phenylindole (DAPI; Invitrogen). .. Mycobacterial protein fragment complementation The system described for investigating protein interactions by the functional reconstitution of a murine dehydrofolate reductase domain in M. tuberculosis [ ] was modified here for use in M. ulcerans.

    Labeling:

    Article Title: Systemic treatment with CAR-engineered T cells against PSCA delays subcutaneous tumor growth and prolongs survival of mice
    Article Snippet: Activated cells were transduced with 50 μl concentrated PSCA-CAR-encoding lentivirus or Mock lentivirus for 4 hours at 37°C in the presence of 10 μg/ml protamine sulphate and 100 IU IL-2 (Sigma-Aldrich). .. For analysis of PSCA-CAR expression, cells were stained with biotinylated protein-L (Genscript, Piscataway, NJ) [ ], washed 3 times with PBS containing 4% BSA, followed by labeling with phycoerythrin (PE)-conjugated streptavidin (BD Biosciences) or stained with Alexa Fluor® 647 F(ab')2 Fragment of Goat Anti-Mouse IgG (H + L) (Invitrogen) and stained with an allophycocyanin (APC)-conjugated anti-CD3 or fluorescein isothiocyanate (FITC)-conjugated anti-CD3 antibody (Nordic Biosite). .. Flow cytometry analysis was performed using FACSCanto II or LSRII (BD Biosciences).

    Incubation:

    Article Title: CaGdt1 plays a compensatory role for the calcium pump CaPmr1 in the regulation of calcium signaling and cell wall integrity signaling in Candida albicans
    Article Snippet: BSA of 400 μg ml− 1 was added to the spheroplast mixture, and incubated for 20 min before mouse anti-HA antibodies was added at a dilution of 1:500. .. The mixture was incubated for 2 h, and washed twice with PBST before goat anti-mouse IgG conjugated to Alexa Fluor 555 (Invitrogen, USA) was added. .. The mixture was incubated in dark for 45 min before spheroplasts were collected, washed three times, and visualized under a Nikon 80i microscope equipped with DS-U2 CCD.

    Article Title: Design of novel small molecule base-pair recognizers of toxic CUG RNA transcripts characteristics of DM1
    Article Snippet: .. Then, samples were incubated with anti-MBNL1 3A4 antibody (Santa Cruz Biotechnology) solution in PBS at room temperature for 1 h. After that samples were washed 3 times with PBS and incubated using goat anti-mouse IgG-Alexa Fluor 488 (Invitrogen, Thermo Scientific, Carlsbad, CA). .. After that, samples were incubated with Hoechst 33,258 (Invitrogen) in PBS at room temperature for 10 min.

    other:

    Article Title: A Soluble Fragment of the Tumor Antigen BCL2-associated Athanogene 6 (BAG-6) Is Essential and Sufficient for Inhibition of NKp30 Receptor-dependent Cytotoxicity of Natural Killer Cells *
    Article Snippet: The following antibodies were used: mouse monoclonal anti-polyhistidine tag HRP-conjugate and anti-human HRP-conjugate (both Sigma-Aldrich), anti-Strep-mAb classic HRP (IBA), anti-NKp30 clone p30-15 (kindly provided by Carsten Watzl), goat anti-mouse IgG1 APC conjugate (Life Technologies), and anti-CD4 APC conjugate (eBioscience).

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  • 94
    Thermo Fisher goat anti mouse igg texas red
    <t>MBL</t> binds to B. burgdorferi , but MBL deficiency in mice does not decrease complement-dependent killing of B. burgdorferi . ( A ) Recombinant human MBL (5 µg/mL) was added to cultured CFSE-labeled B. burgdorferi N40 spirochetes (2 × 10 8 spirochetes/mL) and after two washing steps, binding of MBL to B. burgdorferi was subjected to FACS analysis (red line). As a control, 100 mM EDTA was added together with MBL which abrogates calcium dependent binding of MBL CRD’s (yellow dotted line). The blue line represents spirochetes without MBL. Binding of MBL was determined using a mouse anti-human MBL antibody and a goat anti-mouse <t>IgG</t> Texas Red antibody. ( B ) ELISA assessment of dose-dependent binding of MBL in murine serum to B. burgdorferi membrane protein extract-coated plates. WT serum or MBL deficient murine serum was added to wells and murine MBL was detected using an antibody against murine MBL-A. As a control, normal murine serum was pre-incubated with 100 mM EDTA. ( C ) Growth inhibition of B. burgdorferi strain N40 by murine serum was measured by the color change of Phenol Red at 562/630 nm once a day for up to five days. Ratio NMS/HI NMS and MBL deficient serum/HI MBL deficient serum after five days was 3.66 and 3.01, respectively. ( D ) Growth inhibition of B. garinii strain A87S by murine serum. The ratio NMS/HI NMS and MBL deficient serum/HI MBL deficient serum after five days was 2.84 and 2.86, respectively. ( E ) B. garinii strain A87S was incubated with 25% WT NMS, 25% MBL deficient murine serum or 25% heat-inactivated (HI) NMS (control). After 1 h of incubation the percentages of non-motile spirochetes were determined by a researcher blinded to the experimental design. Each sample and each time point were performed in triplicates and symbols or bars represent the mean ± SEM.
    Goat Anti Mouse Igg Texas Red, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti mouse igg texas red/product/Thermo Fisher
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    86
    Thermo Fisher alexa fluor 488 conjugated goat anti mouse igg antibody
    Chemical structures of the compounds tested and visualization of SARS-CoV-2-infected Vero cells. ( A ) Chemical structures of gemcitabine, 2FdC, and remdesivir. ( B ) MTT-based cytotoxicity assay of gemcitabine at different concentrations and time points. Vero cells were treated with increasing concentrations of gemcitabine for 24 h (black square) or 48 h (gray square). Percentage cell viability was measured by using MTT, in which mock-treated cells served as a control (100%). ( C ) Fluorescein diacetate-based cytotoxicity assay of gemcitabine. Vero cells were treated with increasing concentrations of gemcitabine for 24 (black square) and 48 h (gray square). Percentage cell viability was measured by addition of fluorescein diacetate, in which mock-treated cells served as a control (100%). ( D ) Fluorescein diacetate-based cytotoxicity assay of a delivery vehicle. Increasing concentrations of DMSO, 0.2, 0.6 and 1.8% ( v / v ), that were identically included in 100, 300 and 900 μM gemcitabine shown in ( C ), were treated to Vero cells for 24 (black bar) and 48 h (gray bar). Values in ( B – D ) are means ± standard deviations from thee three independent experiments. ( E ) Visualization of SARS-CoV-2 infection. Vero cells were mock-infected (Mock) or infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.02 for 24 h. Viral spike (S) protein was probed with mouse anti-S antibody and <t>Alexa</t> Fluor 488-conjugated goat anti-mouse antibody (green). Cellular nuclei were counterstained with DAPI (blue). Magnification ×20.
    Alexa Fluor 488 Conjugated Goat Anti Mouse Igg Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 488 conjugated goat anti mouse igg antibody/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
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    95
    Thermo Fisher cross adsorbed goat anti mouse igg conjugated to alexa fluor 568
    Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to <t>Alexa</t> Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa <t>Fluor</t> 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.
    Cross Adsorbed Goat Anti Mouse Igg Conjugated To Alexa Fluor 568, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cross adsorbed goat anti mouse igg conjugated to alexa fluor 568/product/Thermo Fisher
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    MBL binds to B. burgdorferi , but MBL deficiency in mice does not decrease complement-dependent killing of B. burgdorferi . ( A ) Recombinant human MBL (5 µg/mL) was added to cultured CFSE-labeled B. burgdorferi N40 spirochetes (2 × 10 8 spirochetes/mL) and after two washing steps, binding of MBL to B. burgdorferi was subjected to FACS analysis (red line). As a control, 100 mM EDTA was added together with MBL which abrogates calcium dependent binding of MBL CRD’s (yellow dotted line). The blue line represents spirochetes without MBL. Binding of MBL was determined using a mouse anti-human MBL antibody and a goat anti-mouse IgG Texas Red antibody. ( B ) ELISA assessment of dose-dependent binding of MBL in murine serum to B. burgdorferi membrane protein extract-coated plates. WT serum or MBL deficient murine serum was added to wells and murine MBL was detected using an antibody against murine MBL-A. As a control, normal murine serum was pre-incubated with 100 mM EDTA. ( C ) Growth inhibition of B. burgdorferi strain N40 by murine serum was measured by the color change of Phenol Red at 562/630 nm once a day for up to five days. Ratio NMS/HI NMS and MBL deficient serum/HI MBL deficient serum after five days was 3.66 and 3.01, respectively. ( D ) Growth inhibition of B. garinii strain A87S by murine serum. The ratio NMS/HI NMS and MBL deficient serum/HI MBL deficient serum after five days was 2.84 and 2.86, respectively. ( E ) B. garinii strain A87S was incubated with 25% WT NMS, 25% MBL deficient murine serum or 25% heat-inactivated (HI) NMS (control). After 1 h of incubation the percentages of non-motile spirochetes were determined by a researcher blinded to the experimental design. Each sample and each time point were performed in triplicates and symbols or bars represent the mean ± SEM.

    Journal: Scientific Reports

    Article Title: The role of Mannose Binding Lectin in the immune response against Borrelia burgdorferi sensu lato

    doi: 10.1038/s41598-018-37922-8

    Figure Lengend Snippet: MBL binds to B. burgdorferi , but MBL deficiency in mice does not decrease complement-dependent killing of B. burgdorferi . ( A ) Recombinant human MBL (5 µg/mL) was added to cultured CFSE-labeled B. burgdorferi N40 spirochetes (2 × 10 8 spirochetes/mL) and after two washing steps, binding of MBL to B. burgdorferi was subjected to FACS analysis (red line). As a control, 100 mM EDTA was added together with MBL which abrogates calcium dependent binding of MBL CRD’s (yellow dotted line). The blue line represents spirochetes without MBL. Binding of MBL was determined using a mouse anti-human MBL antibody and a goat anti-mouse IgG Texas Red antibody. ( B ) ELISA assessment of dose-dependent binding of MBL in murine serum to B. burgdorferi membrane protein extract-coated plates. WT serum or MBL deficient murine serum was added to wells and murine MBL was detected using an antibody against murine MBL-A. As a control, normal murine serum was pre-incubated with 100 mM EDTA. ( C ) Growth inhibition of B. burgdorferi strain N40 by murine serum was measured by the color change of Phenol Red at 562/630 nm once a day for up to five days. Ratio NMS/HI NMS and MBL deficient serum/HI MBL deficient serum after five days was 3.66 and 3.01, respectively. ( D ) Growth inhibition of B. garinii strain A87S by murine serum. The ratio NMS/HI NMS and MBL deficient serum/HI MBL deficient serum after five days was 2.84 and 2.86, respectively. ( E ) B. garinii strain A87S was incubated with 25% WT NMS, 25% MBL deficient murine serum or 25% heat-inactivated (HI) NMS (control). After 1 h of incubation the percentages of non-motile spirochetes were determined by a researcher blinded to the experimental design. Each sample and each time point were performed in triplicates and symbols or bars represent the mean ± SEM.

    Article Snippet: Spirochetes were washed and bound MBL was detected using mouse anti-human MBL (1:250) (mAb 3E7, Hycult-biotech, Uden, the Netherlands) and goat anti-mouse IgG Texas Red (1:1000, Invitrogen, CA, USA).

    Techniques: Mouse Assay, Recombinant, Cell Culture, Labeling, Binding Assay, FACS, Enzyme-linked Immunosorbent Assay, Incubation, Inhibition

    Increased B. burgdorferi burden in skin tissue but not in other tissues of MBL deficient mice during murine B. burgdorferi infection. ( A – C ) WT and MBL deficient mice (8 per group) were infected with 10 6 B. burgdorferi N40 via subcutaneous inoculation. Mice were sacrificed after 14 days and assessed for B. burgdorferi burden. DNA was extracted using the Qiagen Blood and Tissue kit from skin, heart, joint and bladder and were subjected to quantitative B. burgdorferi flaB PCR normalized for quantitative mouse β-actin PCR. A. qPCR assessment of B. burgdorferi burden in skin after 14 days. ( B ) IgG and IgM titers against B. burgdorferi lysate in serum measured by ELISA. ( C ) qPCR assessment of B. burgdorferi burden in joint, bladder or heart. ( D,E ) WT and MBL deficient mice (8 per group) were infected with B. burgdorferi N40 via a tick challenge (4–5 ticks/mouse). Two separate experiments were performed, one with three animals per group and one with five animals per group. B. burgdorferi burden was normalized to the highest WT control value per experiment to pool both experiments. ( D ) qPCR assessment of B. burgdorferi burden in skin from ear biopsy at 7 days. ( E ) qPCR assessment of B. burgdorferi burden in skin, joint, bladder or heart at 21 days. ( F,G ) Five µm-thick sagittal heart sections were stained with hematoxylin and eosin. A carditis score was performed by an independent pathologist who was blinded to the experimental design on a scale from 0–3 with 0 being no, 1 mild, 2 moderate, and 3 being severe carditis. Carditis was characterized by disperse inflammation at the atrioventricular junction and aortic root. See Fig. S1 for a normal heart from a mice injected with PBS as a control. Error bars represent mean ± SEM and mean values significantly different in a two-tailed non-parametric Mann-Whitney test are indicated by asterisks (*p

    Journal: Scientific Reports

    Article Title: The role of Mannose Binding Lectin in the immune response against Borrelia burgdorferi sensu lato

    doi: 10.1038/s41598-018-37922-8

    Figure Lengend Snippet: Increased B. burgdorferi burden in skin tissue but not in other tissues of MBL deficient mice during murine B. burgdorferi infection. ( A – C ) WT and MBL deficient mice (8 per group) were infected with 10 6 B. burgdorferi N40 via subcutaneous inoculation. Mice were sacrificed after 14 days and assessed for B. burgdorferi burden. DNA was extracted using the Qiagen Blood and Tissue kit from skin, heart, joint and bladder and were subjected to quantitative B. burgdorferi flaB PCR normalized for quantitative mouse β-actin PCR. A. qPCR assessment of B. burgdorferi burden in skin after 14 days. ( B ) IgG and IgM titers against B. burgdorferi lysate in serum measured by ELISA. ( C ) qPCR assessment of B. burgdorferi burden in joint, bladder or heart. ( D,E ) WT and MBL deficient mice (8 per group) were infected with B. burgdorferi N40 via a tick challenge (4–5 ticks/mouse). Two separate experiments were performed, one with three animals per group and one with five animals per group. B. burgdorferi burden was normalized to the highest WT control value per experiment to pool both experiments. ( D ) qPCR assessment of B. burgdorferi burden in skin from ear biopsy at 7 days. ( E ) qPCR assessment of B. burgdorferi burden in skin, joint, bladder or heart at 21 days. ( F,G ) Five µm-thick sagittal heart sections were stained with hematoxylin and eosin. A carditis score was performed by an independent pathologist who was blinded to the experimental design on a scale from 0–3 with 0 being no, 1 mild, 2 moderate, and 3 being severe carditis. Carditis was characterized by disperse inflammation at the atrioventricular junction and aortic root. See Fig. S1 for a normal heart from a mice injected with PBS as a control. Error bars represent mean ± SEM and mean values significantly different in a two-tailed non-parametric Mann-Whitney test are indicated by asterisks (*p

    Article Snippet: Spirochetes were washed and bound MBL was detected using mouse anti-human MBL (1:250) (mAb 3E7, Hycult-biotech, Uden, the Netherlands) and goat anti-mouse IgG Texas Red (1:1000, Invitrogen, CA, USA).

    Techniques: Mouse Assay, Infection, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Staining, Injection, Two Tailed Test, MANN-WHITNEY

    FLNa-EGFP colocalizes with actin at the membrane and in fibres. ( A ) Confocal images of FLNa, FLNa-EGFP and actin expression in cells after immunostaining. M2 cells transiently transfected with pcDNA3.1-FLNa-EGFP or pREP4-FLNa and untransfected A7 and M2 cells, were seeded on top of glass coverslips, fixed, permeabilized, incubated with anti-FLNa and goat anti-mouse Alexa Fluor 488 and stained with rhodamine-phalloidin before mounting. ( B ) Confocal images of FLNa-EGFP expression in M2 cells transiently transfected with pcDNA3.1-FLNa-EGFP. Cells were seeded on top of glass coverslips, fixed, permeabilized and incubated with anti-FLNa and Texas Red goat anti-mouse. All images are from one single layer of the Z stacks. The colocalization between (green) and (red) was analyzed using Imaris colocalization software and is shown in white. Images are representative of at least three independent experiments. Scalebars (10 μm).

    Journal: PLoS ONE

    Article Title: Filamin A-Hinge Region 1-EGFP: A Novel Tool for Tracking the Cellular Functions of Filamin A in Real-Time

    doi: 10.1371/journal.pone.0040864

    Figure Lengend Snippet: FLNa-EGFP colocalizes with actin at the membrane and in fibres. ( A ) Confocal images of FLNa, FLNa-EGFP and actin expression in cells after immunostaining. M2 cells transiently transfected with pcDNA3.1-FLNa-EGFP or pREP4-FLNa and untransfected A7 and M2 cells, were seeded on top of glass coverslips, fixed, permeabilized, incubated with anti-FLNa and goat anti-mouse Alexa Fluor 488 and stained with rhodamine-phalloidin before mounting. ( B ) Confocal images of FLNa-EGFP expression in M2 cells transiently transfected with pcDNA3.1-FLNa-EGFP. Cells were seeded on top of glass coverslips, fixed, permeabilized and incubated with anti-FLNa and Texas Red goat anti-mouse. All images are from one single layer of the Z stacks. The colocalization between (green) and (red) was analyzed using Imaris colocalization software and is shown in white. Images are representative of at least three independent experiments. Scalebars (10 μm).

    Article Snippet: Cells were then washed in PBS, fixed and permeabilized in PBS with 0.1% Tween 20 before staining with mouse anti-filamin 1 (Santa Cruz Biotechnologies, CA, USA), Alexa Fluor 488 goat anti-mouse (Invitrogen, CA, USA) or Texas Red goat anti-mouse (Invitrogen, CA, USA) and Rhodamine-Phalloidin (Invitrogen, CA, USA).

    Techniques: Expressing, Immunostaining, Transfection, Incubation, Staining, Software

    Chemical structures of the compounds tested and visualization of SARS-CoV-2-infected Vero cells. ( A ) Chemical structures of gemcitabine, 2FdC, and remdesivir. ( B ) MTT-based cytotoxicity assay of gemcitabine at different concentrations and time points. Vero cells were treated with increasing concentrations of gemcitabine for 24 h (black square) or 48 h (gray square). Percentage cell viability was measured by using MTT, in which mock-treated cells served as a control (100%). ( C ) Fluorescein diacetate-based cytotoxicity assay of gemcitabine. Vero cells were treated with increasing concentrations of gemcitabine for 24 (black square) and 48 h (gray square). Percentage cell viability was measured by addition of fluorescein diacetate, in which mock-treated cells served as a control (100%). ( D ) Fluorescein diacetate-based cytotoxicity assay of a delivery vehicle. Increasing concentrations of DMSO, 0.2, 0.6 and 1.8% ( v / v ), that were identically included in 100, 300 and 900 μM gemcitabine shown in ( C ), were treated to Vero cells for 24 (black bar) and 48 h (gray bar). Values in ( B – D ) are means ± standard deviations from thee three independent experiments. ( E ) Visualization of SARS-CoV-2 infection. Vero cells were mock-infected (Mock) or infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.02 for 24 h. Viral spike (S) protein was probed with mouse anti-S antibody and Alexa Fluor 488-conjugated goat anti-mouse antibody (green). Cellular nuclei were counterstained with DAPI (blue). Magnification ×20.

    Journal: International Journal of Molecular Sciences

    Article Title: Comparison of Antiviral Activity of Gemcitabine with 2′-Fluoro-2′-Deoxycytidine and Combination Therapy with Remdesivir against SARS-CoV-2

    doi: 10.3390/ijms22041581

    Figure Lengend Snippet: Chemical structures of the compounds tested and visualization of SARS-CoV-2-infected Vero cells. ( A ) Chemical structures of gemcitabine, 2FdC, and remdesivir. ( B ) MTT-based cytotoxicity assay of gemcitabine at different concentrations and time points. Vero cells were treated with increasing concentrations of gemcitabine for 24 h (black square) or 48 h (gray square). Percentage cell viability was measured by using MTT, in which mock-treated cells served as a control (100%). ( C ) Fluorescein diacetate-based cytotoxicity assay of gemcitabine. Vero cells were treated with increasing concentrations of gemcitabine for 24 (black square) and 48 h (gray square). Percentage cell viability was measured by addition of fluorescein diacetate, in which mock-treated cells served as a control (100%). ( D ) Fluorescein diacetate-based cytotoxicity assay of a delivery vehicle. Increasing concentrations of DMSO, 0.2, 0.6 and 1.8% ( v / v ), that were identically included in 100, 300 and 900 μM gemcitabine shown in ( C ), were treated to Vero cells for 24 (black bar) and 48 h (gray bar). Values in ( B – D ) are means ± standard deviations from thee three independent experiments. ( E ) Visualization of SARS-CoV-2 infection. Vero cells were mock-infected (Mock) or infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.02 for 24 h. Viral spike (S) protein was probed with mouse anti-S antibody and Alexa Fluor 488-conjugated goat anti-mouse antibody (green). Cellular nuclei were counterstained with DAPI (blue). Magnification ×20.

    Article Snippet: Viral S protein was probed using anti-S antibody (Cat. No., GTX632604; Genetex, Irvine, CA, USA) and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen, Carlsbad, CA, USA), while cellular nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen).

    Techniques: Infection, MTT Assay, Cytotoxicity Assay

    Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to Alexa Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Journal: Infection and Immunity

    Article Title: Internalization and Trafficking of Nontypeable Haemophilus influenzae in Human Respiratory Epithelial Cells and Roles of IgA1 Proteases for Optimal Invasion and Persistence

    doi: 10.1128/IAI.00864-13

    Figure Lengend Snippet: Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to Alexa Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Article Snippet: Host cell components were visualized using filipin III fluorescent dye (final concentration of 333 μg/ml; Cayman Chemical), primary monoclonal antibodies including mouse anti-caveolin-1 antibody (1:50; BD), mouse anti-early endosomal antigen 1 (EEA-1) antibody (1:200; BD), mouse anti-flotillin-1 antibody (1:20; BD), mouse anti-human CD107a antibody (LAMP1) (1:200; BD), and goat anti-human secretory component antibody (1:200; Sigma), and secondary antibodies including highly cross-adsorbed goat anti-mouse IgG conjugated to Alexa Fluor 568 (Life Technologies, 1:200) and donkey anti-goat IgG conjugated to Alexa Fluor 568 (Life Technologies, 1:200).

    Techniques: Confocal Microscopy, Labeling, Incubation, Infection, Invasion Assay

    Confocal microscopy and survival assay with pharmacologic inhibitor of lysosome acidification. NTHI traffics to early endosomes and lysosomes and is killed in lysosomes. IgA1 proteases are required for optimal survival in lysosomes. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI is found within vesicles positive for EEA1 (shown at 4 h) and within vesicles positive for LAMP1 (shown at 24 h). NTHI was labeled using anti-11P6H antibody conjugated to Alexa Fluor 488 (green). EEA1 was labeled using anti-EEA1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). LAMP1 was labeled using anti-human LAMP1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). (B) Survival assay in the presence and absence of concanamycin A (CMA). H292 cells were infected for 16 h, treated with gentamicin for 1 h, and treated with concanamycin or diluent for 3 h. Survival data are normalized to samples treated with diluent. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Journal: Infection and Immunity

    Article Title: Internalization and Trafficking of Nontypeable Haemophilus influenzae in Human Respiratory Epithelial Cells and Roles of IgA1 Proteases for Optimal Invasion and Persistence

    doi: 10.1128/IAI.00864-13

    Figure Lengend Snippet: Confocal microscopy and survival assay with pharmacologic inhibitor of lysosome acidification. NTHI traffics to early endosomes and lysosomes and is killed in lysosomes. IgA1 proteases are required for optimal survival in lysosomes. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI is found within vesicles positive for EEA1 (shown at 4 h) and within vesicles positive for LAMP1 (shown at 24 h). NTHI was labeled using anti-11P6H antibody conjugated to Alexa Fluor 488 (green). EEA1 was labeled using anti-EEA1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). LAMP1 was labeled using anti-human LAMP1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). (B) Survival assay in the presence and absence of concanamycin A (CMA). H292 cells were infected for 16 h, treated with gentamicin for 1 h, and treated with concanamycin or diluent for 3 h. Survival data are normalized to samples treated with diluent. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Article Snippet: Host cell components were visualized using filipin III fluorescent dye (final concentration of 333 μg/ml; Cayman Chemical), primary monoclonal antibodies including mouse anti-caveolin-1 antibody (1:50; BD), mouse anti-early endosomal antigen 1 (EEA-1) antibody (1:200; BD), mouse anti-flotillin-1 antibody (1:20; BD), mouse anti-human CD107a antibody (LAMP1) (1:200; BD), and goat anti-human secretory component antibody (1:200; Sigma), and secondary antibodies including highly cross-adsorbed goat anti-mouse IgG conjugated to Alexa Fluor 568 (Life Technologies, 1:200) and donkey anti-goat IgG conjugated to Alexa Fluor 568 (Life Technologies, 1:200).

    Techniques: Confocal Microscopy, Clonogenic Cell Survival Assay, Labeling, Infection