whole goat immunoglobulin g igg  (Jackson Immuno)

 
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    Name:
    Cy 3 AffiniPure Goat Anti Mouse IgG subclasses 1 2a 2b 3 Fcγ Fragment Specific
    Description:
    Whole IgG antibodies are isolated as intact molecules from antisera by immunoaffinity chromatography They have an Fc portion and two antigen binding Fab portions joined together by disulfide bonds and therefore they are divalent The average molecular weight is reported to be about 160 kDa The whole IgG form of antibodies is suitable for the majority of immunodetection procedures and is the most cost effective Based on immunoelectrophoresis and or ELISA the antibody reacts with mouse IgG subclasses 1 2a 2b and 3 but not with the Fab portion of mouse immunoglobulins No antibody was detected against mouse IgM or non immunoglobulin serum proteins The antibody has been tested by ELISA and or solid phase adsorbed to ensure minimal cross reaction with human bovine and rabbit serum proteins but it may cross react with immunoglobulins from other species
    Catalog Number:
    115-165-164
    Price:
    193.0
    Category:
    Whole IgG Affinity Purified Antibodies
    Conjugate:
    Cyanine Cy 3
    Size:
    1 0 mg
    Format:
    Whole IgG
    Host:
    Goat
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    Structured Review

    Jackson Immuno whole goat immunoglobulin g igg
    BRV p15 is targeted to the endoplasmic reticulum. (A) Transfected cells expressing p15 were incubated in the absence or presence of brefeldin A, as outlined in Materials and Methods. The extent of syncytium formation was determined by Wright-Giemsa staining to reveal the presence of clustered nuclei in polykaryons, and images were captured by light microscopy at ×170 magnification. (B) Transfected cells expressing p15 were methanol-fixed and coimmunostained using rabbit anti-p15 polyclonal antiserum and FITC-conjugated goat anti-rabbit F(ab′)2 (α-p15 panel), followed by mouse anti-calnexin and rhodamine-conjugated goat anti-mouse <t>IgG</t> (α-calnexin panel). The bottom panel shows a merge of the images in the first two panels, with colocalization indicated by the yellow color. Scale bar, 10 μm.
    Whole IgG antibodies are isolated as intact molecules from antisera by immunoaffinity chromatography They have an Fc portion and two antigen binding Fab portions joined together by disulfide bonds and therefore they are divalent The average molecular weight is reported to be about 160 kDa The whole IgG form of antibodies is suitable for the majority of immunodetection procedures and is the most cost effective Based on immunoelectrophoresis and or ELISA the antibody reacts with mouse IgG subclasses 1 2a 2b and 3 but not with the Fab portion of mouse immunoglobulins No antibody was detected against mouse IgM or non immunoglobulin serum proteins The antibody has been tested by ELISA and or solid phase adsorbed to ensure minimal cross reaction with human bovine and rabbit serum proteins but it may cross react with immunoglobulins from other species
    https://www.bioz.com/result/whole goat immunoglobulin g igg/product/Jackson Immuno
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    whole goat immunoglobulin g igg - by Bioz Stars, 2021-05
    97/100 stars

    Images

    1) Product Images from "Unusual Topological Arrangement of Structural Motifs in the Baboon Reovirus Fusion-Associated Small Transmembrane Protein"

    Article Title: Unusual Topological Arrangement of Structural Motifs in the Baboon Reovirus Fusion-Associated Small Transmembrane Protein

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.10.6216-6226.2005

    BRV p15 is targeted to the endoplasmic reticulum. (A) Transfected cells expressing p15 were incubated in the absence or presence of brefeldin A, as outlined in Materials and Methods. The extent of syncytium formation was determined by Wright-Giemsa staining to reveal the presence of clustered nuclei in polykaryons, and images were captured by light microscopy at ×170 magnification. (B) Transfected cells expressing p15 were methanol-fixed and coimmunostained using rabbit anti-p15 polyclonal antiserum and FITC-conjugated goat anti-rabbit F(ab′)2 (α-p15 panel), followed by mouse anti-calnexin and rhodamine-conjugated goat anti-mouse IgG (α-calnexin panel). The bottom panel shows a merge of the images in the first two panels, with colocalization indicated by the yellow color. Scale bar, 10 μm.
    Figure Legend Snippet: BRV p15 is targeted to the endoplasmic reticulum. (A) Transfected cells expressing p15 were incubated in the absence or presence of brefeldin A, as outlined in Materials and Methods. The extent of syncytium formation was determined by Wright-Giemsa staining to reveal the presence of clustered nuclei in polykaryons, and images were captured by light microscopy at ×170 magnification. (B) Transfected cells expressing p15 were methanol-fixed and coimmunostained using rabbit anti-p15 polyclonal antiserum and FITC-conjugated goat anti-rabbit F(ab′)2 (α-p15 panel), followed by mouse anti-calnexin and rhodamine-conjugated goat anti-mouse IgG (α-calnexin panel). The bottom panel shows a merge of the images in the first two panels, with colocalization indicated by the yellow color. Scale bar, 10 μm.

    Techniques Used: Transfection, Expressing, Incubation, Staining, Light Microscopy

    Related Articles

    Incubation:

    Article Title: Mouse IgM Fc receptor, FCMR, promotes B cell development and modulates antigen-driven immune responses
    Article Snippet: Aliquots of 1.25–5.0 × 105 spleen and BM cells were plated in duplicate in NP-BSA pre-coated 96-well PVDF membrane plates (Millipore) and were incubated for 1 day at 37°C in 5% CO2 . .. The plates were washed with PBS containing 0.05% Tween-20 and incubated with HRP-conjugated anti-mouse IgM or IgG1 (Jackson ImmunoResearch Laboratory), followed by reaction with FAST 5-bromo-4-chlor-3-indolyl phosphate/NBT chromogen substrate (Sigma). .. The spots were detected with a CTL-ImmunoSpot® S5 Core Analyzer (Cellular Technology) and analyzed by ImmunoSpot® Software 4.0 (Cellular Technology).

    Staining:

    Article Title: Blimp-1 controls plasma cell function through regulation of immunoglobulin secretion and the unfolded protein response
    Article Snippet: Cells were stained with lysotracker deep red or ERtracker red (Molecular Probes) according to manufacturer’s instructions. .. For intracellular transcription factor and immunoglobulin measurement, cells were fixed and permeabilized using the eBiosciences transcription factor staining buffer set and BD Cytofix/Cytoperm kit respectively, then stained antibodies specific for: XBP-1s (Q3-695; BD Biosciences), ATF4 (D4B8; Cell Signaling), ATF6 (polyclonal; Abcam), IgM (polyclonal; Jackson Immunoresearch), IgG (polyclonal; Jackson Immunoresearch), IgA (mA-6E1; eBiosciences), Igκ (187.1; BD Biosciences), Igλ (TB28-2; BD biosciences). .. For phospho-protein detection, cells were prepared using BD Phosphoflow lyse/fix buffer and perm buffer III, and labeled with specific antibodies that recognize: p-S235/236-S6 (D57.2.25; Cell Signaling), p-S2448-mTOR (D9C2; Cell Signaling), p-S79-ACC (D7D11; Cell Signaling), p-S792-Raptor (polyclonal: Cell Signaling), p-S473-Akt (M89-61, BD biosciences).

    Infection:

    Article Title: Protective Immunity against Recurrent Staphylococcus aureus Skin Infection Requires Antibody and Interleukin-17A
    Article Snippet: Similarly, total anti-Hla IgG levels did not differ between the two backgrounds after primary infection ( P = 0.4) ( ). .. In contrast, there were higher levels of anti-Hla total IgG, IgG1, IgG2a, and IgG3 ( P < 0.01) but not IgG2b after secondary infection of BALB/c mice, compared with results for C57BL/6 mice ( and ). .. These data suggested that a selective difference in the antibody response in BALB/c mice from that in C57BL/6 mice might be the basis for differential antibody-mediated protection.

    Mouse Assay:

    Article Title: Protective Immunity against Recurrent Staphylococcus aureus Skin Infection Requires Antibody and Interleukin-17A
    Article Snippet: Similarly, total anti-Hla IgG levels did not differ between the two backgrounds after primary infection ( P = 0.4) ( ). .. In contrast, there were higher levels of anti-Hla total IgG, IgG1, IgG2a, and IgG3 ( P < 0.01) but not IgG2b after secondary infection of BALB/c mice, compared with results for C57BL/6 mice ( and ). .. These data suggested that a selective difference in the antibody response in BALB/c mice from that in C57BL/6 mice might be the basis for differential antibody-mediated protection.

    Labeling:

    Article Title: Distinctive Morphological Features of a Subset of Cortical Neurons Grown in the Presence of Basal Forebrain Neurons In Vitro
    Article Snippet: For AChE and SMI-32 double labeling, the AChE histochemistry was performed first, followed by SMI-32 immunocytochemistry. .. Again, an anti-mouse IgG–Cy3 secondary antibody was used to visualize cells labeled with the SMI-32 primary antibody. .. Again, an anti-mouse IgG–Cy3 secondary antibody was used to visualize cells labeled with the SMI-32 primary antibody.

    Generated:

    Article Title: Evaluation of Virus-Like Particle-Based Tumor-Associated Carbohydrate Immunogen in a Mouse Tumor Model
    Article Snippet: The plate is incubated for 2h at 37°C and then washed with PBST (4×200μL). .. A 1:2000 dilution of HRP-conjugated goat antimouse IgG, IgM, IgG1, IgG2b, IgG2c, or IgG3 (Jackson ImmunoResearch Laboratory) in 0.1% BSA/PBS (100μL) is added to the wells, respectively, to determine the subtypes of antibodies generated. ..

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  • 94
    Jackson Immuno goat anti mouse immunoglobulin g igg
    Killing of wild-type and mutant strains of M. catarrhalis by human sera. (A) Effect of various sera on selected strains. The wild-type strain O35E (1), the uspA2 mutant O35EΔ2 (2), and the wild-type strain MC317 (3) were incubated for 30 min at 37°C in VBS ++ containing 10% (vol/vol) heat-inactivated NHS (a), 10% (vol/vol) NHS (b), or 10% (vol/vol) factor B-depleted human serum (c) or in VBS containing 10 mM MgCl 2 , 10 mM EGTA, and 10% (vol/vol) NHS (d). Portions of these reaction mixtures were plated at time zero and after 30 min, and the percentage of survival is calculated relative to the original inoculum. The data presented here are the means of results from three independent experiments plus the standard errors. (B) Involvement of <t>IgG</t> in killing of the uspA2 mutant O35EΔ2. Wild-type strain O35E (1) and the uspA2 mutant O35EΔ2 (2) were incubated with the following sera: 10% (vol/vol) heat-inactivated NHS (a), 10% (vol/vol) NHS (b), 10% (vol/vol) IgG-depleted NHS (c), and 10% (vol/vol) IgG-depleted NHS mixed with heat-inactivated NHS as a source of IgG (d). The data presented here are the means of results from three independent experiments plus the standard errors.
    Goat Anti Mouse Immunoglobulin G Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    95
    Jackson Immuno phycoerythrin pe conjugated anti mouse immunoglobulin g
    Antibody-mediated inhibition of cellular HCV-LP binding. HCV-LPs were incubated with anti-HCV-positive serum or a pool of anti-HCV-negative control sera (dilution, 1:50). HCV-LP-antibody complexes were added to HuH-7 cells for 1 h at 4°C. After removal of nonbound HCV-LP-antibody complexes by washing the cells in PBS-2% BSA, the binding of HCV-LPs was detected by flow cytometry using the mouse monoclonal anti-E2 antibody AP33 and PE-conjugated anti-mouse <t>IgG</t> (A and B) or human polyclonal anti-HCV and FITC-conjugated anti-human IgG antibody (C). The fluorescence intensity and relative cell numbers (Counts) are shown on the x and y axes, respectively. NC, negative control, corresponding to HuH-7 cells incubated with control insect cell preparations (GUS) and control serum. (D) For the assessment of concentration-dependent antibody-mediated inhibition of binding, HCV-LPs (genotype 1b) were incubated in subsaturating concentrations with an anti-HCV-positive serum or a pool of anti-HCV-negative control sera (at various dilutions as indicated on the x axis) for 1 h at 37°C. HCV-LP-antibody complexes were added to HuH-7 cells for 1 h at 4°C. HCV-LP binding to the HuH-7 cells was detected by flow cytometry as described above. Inhibition of cellular HCV-LP binding ( y axis) was calculated as described in Materials and Methods.
    Phycoerythrin Pe Conjugated Anti Mouse Immunoglobulin G, supplied by Jackson Immuno, 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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    90
    Jackson Immuno hamster immunoglobulin g
    Th differentiation and B cell responses in mice deprived of functional RLTPR molecules. (A) Sorted naive CD4 + T cells (2 × 10 5 ) from mice of the specified genotype were stimulated for 5 d with anti-CD3 and -CD28 under Th1, Th2, or Th17 differentiating conditions. After 5 d of culture, the absolute number of IFN-γ + (Th1 condition), IL-4 + (Th2 condition), and IL-17 + (Th17 condition) CD4 + T cells was determined. Each dot corresponds to a mouse and the mean (horizontal bar) is indicated. (B) WT and Rltpr bas/bas mice were immunized intraperitoneally at day 0 and 14 with the T cell–dependent antigen TNP-KLH. The concentration of TNP-specific immunoglobulins of the indicated isotypes <t>(IgG2a,</t> <t>IgG2b,</t> and <t>IgG1)</t> were assessed in individual mice before and 21 d after immunization. (C) WT and Rltpr bas/bas mice were immunized with the T cell–independent antigen TNP-LPS, and the concentration of TNP-specific IgM was assessed in individual mice before and 7 d after immunization. (D) Splenic B cells from mice of the specified genotype were stimulated with F(ab)’ 2 goat anti–mouse IgM antibody in the presence or absence of anti-CD40 antibody, or LPS. After 4 d of culture, B cell proliferation was evaluated. Mean and SEM are shown. Data are representative of two independent experiments. In A–C, each dot corresponds to a mouse and the mean (horizontal bar) is indicated. **, P ≤ 0.01; ****, P ≤ 0.001; ns, nonsignificant. In D, two animals were used per genotype.
    Hamster Immunoglobulin G, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Jackson Immuno anti goat horseradish peroxidase conjugated immunoglobulin g igg
    (A,B) Lactoferrin (Lf) fragments accumulate in macrophages following incubation with senescent neutrophils. (A) Peritoneal macrophages were recovered 66 h post zymosan A-induced (1 mg/mouse) peritonitis initiation and incubated with apoptotic Jurkat cells (AC), senescent peritoneal neutrophils (SN), latex beads (LBs), <t>IgG-opsonized</t> LB (oLB), or anti-CD11b monoclonal antibodies as indicated. After 24 h, unbound cells were washed and macrophages were recovered. Then, the cells were lysed, and equal amounts of protein extract were blotted for Lf. Protein extracts from apoptotic neutrophils were also analyzed as indicated. Results are representative from three independent experiments (cells were pooled from three to five mice). (B) Peritoneal neutrophils were recovered 24 h PPI and treated with roscovitine (10 µM) alone or with protease inhibitor cocktail (Pi), cycloheximide (CHX), or the neutrophil elastase inhibitor, silevestat (NEi) for 20 h. Then, neutrophils were lysed, and their protein content was immunoblotted for Lf [results are representative from three experiments (cells were pooled from three to five mice)]. (C,D) Lf fragments are found in interstitial fluids (ISFs) of spleen and inguinal lymph nodes (LN) during peritonitis. Spleen (C) and inguinal LN (D) were harvested 24 and 66 h after initiation of peritonitis ( n = 4–5) and mashed in equal volumes of PBS employing a nylon grid. Equal amounts of ISF protein were run by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), followed by Western blotting for Lf. Results are representative blots from three experiments. (E–G) Peritoneal resolution phase macrophages are essential for the differential production of splenic Lf fragments. Mice were injected intraperitoneally (i.p.) with clodronate-containing or empty liposomes 42 h PPI or before the initiation of peritonitis. After additional 24 h, the spleens were recovered and mashed in equal volumes of PBS employing a nylon grid. Equal amounts of ISF protein were run by 12% SDS-PAGE, followed by Western blotting for Lf. Results are a representative blot (E) and averages of densitometric analysis of the 23- (F) and 17 (G) -kDa bands from three experiments ( n = 3–6 mice per group). **/*indicate statistically significant differences of P ≤ 0.01/ P ≤ 0.05, respectively, by ANOVA with Tukey post hoc analysis. (H) Lf fragments are found in dairy cow milk during the onset and resolution of mastitis. Milk samples from dairy cows with mastitis (days 1–7) were defatted and separated into soluble and cellular fractions by centrifugation. The cellular fraction was lysed and the cytoplasmic proteins were recovered. Equal amounts of protein from both fractions were analyzed by Western blotting for Lf. Results show representative blots from three sample sets taken daily from three different cows.
    Anti Goat Horseradish Peroxidase Conjugated Immunoglobulin G Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Killing of wild-type and mutant strains of M. catarrhalis by human sera. (A) Effect of various sera on selected strains. The wild-type strain O35E (1), the uspA2 mutant O35EΔ2 (2), and the wild-type strain MC317 (3) were incubated for 30 min at 37°C in VBS ++ containing 10% (vol/vol) heat-inactivated NHS (a), 10% (vol/vol) NHS (b), or 10% (vol/vol) factor B-depleted human serum (c) or in VBS containing 10 mM MgCl 2 , 10 mM EGTA, and 10% (vol/vol) NHS (d). Portions of these reaction mixtures were plated at time zero and after 30 min, and the percentage of survival is calculated relative to the original inoculum. The data presented here are the means of results from three independent experiments plus the standard errors. (B) Involvement of IgG in killing of the uspA2 mutant O35EΔ2. Wild-type strain O35E (1) and the uspA2 mutant O35EΔ2 (2) were incubated with the following sera: 10% (vol/vol) heat-inactivated NHS (a), 10% (vol/vol) NHS (b), 10% (vol/vol) IgG-depleted NHS (c), and 10% (vol/vol) IgG-depleted NHS mixed with heat-inactivated NHS as a source of IgG (d). The data presented here are the means of results from three independent experiments plus the standard errors.

    Journal: Infection and Immunity

    Article Title: The UspA2 Protein of Moraxella catarrhalis Is Directly Involved in the Expression of Serum Resistance

    doi: 10.1128/IAI.73.4.2400-2410.2005

    Figure Lengend Snippet: Killing of wild-type and mutant strains of M. catarrhalis by human sera. (A) Effect of various sera on selected strains. The wild-type strain O35E (1), the uspA2 mutant O35EΔ2 (2), and the wild-type strain MC317 (3) were incubated for 30 min at 37°C in VBS ++ containing 10% (vol/vol) heat-inactivated NHS (a), 10% (vol/vol) NHS (b), or 10% (vol/vol) factor B-depleted human serum (c) or in VBS containing 10 mM MgCl 2 , 10 mM EGTA, and 10% (vol/vol) NHS (d). Portions of these reaction mixtures were plated at time zero and after 30 min, and the percentage of survival is calculated relative to the original inoculum. The data presented here are the means of results from three independent experiments plus the standard errors. (B) Involvement of IgG in killing of the uspA2 mutant O35EΔ2. Wild-type strain O35E (1) and the uspA2 mutant O35EΔ2 (2) were incubated with the following sera: 10% (vol/vol) heat-inactivated NHS (a), 10% (vol/vol) NHS (b), 10% (vol/vol) IgG-depleted NHS (c), and 10% (vol/vol) IgG-depleted NHS mixed with heat-inactivated NHS as a source of IgG (d). The data presented here are the means of results from three independent experiments plus the standard errors.

    Article Snippet: The secondary antibody used for Western blot analysis was goat anti-mouse immunoglobulin G (IgG) conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, Pa.).

    Techniques: Mutagenesis, Incubation

    Comparison of UspA2 proteins from M. catarrhalis strains O35E and MC317. (A) Alignment of the deduced amino acid sequences. Identical amino acids are shaded in dark gray, while conserved amino acids are shaded with light gray. This figure was generated by using the ClustalW Alignment program in MacVector (version 6.5). (B) Relative amounts of UspA2 exposed on the surface of the following strains as measured by the indirect antibody accessibility assay: the M. catarrhalis uspA1 uspA2 mutant O35E.12 (1), the uspA1 mutant O35E.1 (2), and the uspA1 mutant MC317.1 (3). The counts per minute of radioiodinated goat anti-mouse IgG bound to MAb 17C7 on the bacterial cell surface (a) are plotted on the y axis. MAb 3F12, a murine IgG MAb specific for the major outer membrane protein of H. ducreyi ), was used as the negative control (b). These data represent the means of results from two independent experiments plus standard deviations.

    Journal: Infection and Immunity

    Article Title: The UspA2 Protein of Moraxella catarrhalis Is Directly Involved in the Expression of Serum Resistance

    doi: 10.1128/IAI.73.4.2400-2410.2005

    Figure Lengend Snippet: Comparison of UspA2 proteins from M. catarrhalis strains O35E and MC317. (A) Alignment of the deduced amino acid sequences. Identical amino acids are shaded in dark gray, while conserved amino acids are shaded with light gray. This figure was generated by using the ClustalW Alignment program in MacVector (version 6.5). (B) Relative amounts of UspA2 exposed on the surface of the following strains as measured by the indirect antibody accessibility assay: the M. catarrhalis uspA1 uspA2 mutant O35E.12 (1), the uspA1 mutant O35E.1 (2), and the uspA1 mutant MC317.1 (3). The counts per minute of radioiodinated goat anti-mouse IgG bound to MAb 17C7 on the bacterial cell surface (a) are plotted on the y axis. MAb 3F12, a murine IgG MAb specific for the major outer membrane protein of H. ducreyi ), was used as the negative control (b). These data represent the means of results from two independent experiments plus standard deviations.

    Article Snippet: The secondary antibody used for Western blot analysis was goat anti-mouse immunoglobulin G (IgG) conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, Pa.).

    Techniques: Generated, Mutagenesis, Negative Control

    Antibody-mediated inhibition of cellular HCV-LP binding. HCV-LPs were incubated with anti-HCV-positive serum or a pool of anti-HCV-negative control sera (dilution, 1:50). HCV-LP-antibody complexes were added to HuH-7 cells for 1 h at 4°C. After removal of nonbound HCV-LP-antibody complexes by washing the cells in PBS-2% BSA, the binding of HCV-LPs was detected by flow cytometry using the mouse monoclonal anti-E2 antibody AP33 and PE-conjugated anti-mouse IgG (A and B) or human polyclonal anti-HCV and FITC-conjugated anti-human IgG antibody (C). The fluorescence intensity and relative cell numbers (Counts) are shown on the x and y axes, respectively. NC, negative control, corresponding to HuH-7 cells incubated with control insect cell preparations (GUS) and control serum. (D) For the assessment of concentration-dependent antibody-mediated inhibition of binding, HCV-LPs (genotype 1b) were incubated in subsaturating concentrations with an anti-HCV-positive serum or a pool of anti-HCV-negative control sera (at various dilutions as indicated on the x axis) for 1 h at 37°C. HCV-LP-antibody complexes were added to HuH-7 cells for 1 h at 4°C. HCV-LP binding to the HuH-7 cells was detected by flow cytometry as described above. Inhibition of cellular HCV-LP binding ( y axis) was calculated as described in Materials and Methods.

    Journal: Journal of Virology

    Article Title: Inhibition of Hepatitis C Virus-Like Particle Binding to Target Cells by Antiviral Antibodies in Acute and Chronic Hepatitis C

    doi: 10.1128/JVI.78.17.9030-9040.2004

    Figure Lengend Snippet: Antibody-mediated inhibition of cellular HCV-LP binding. HCV-LPs were incubated with anti-HCV-positive serum or a pool of anti-HCV-negative control sera (dilution, 1:50). HCV-LP-antibody complexes were added to HuH-7 cells for 1 h at 4°C. After removal of nonbound HCV-LP-antibody complexes by washing the cells in PBS-2% BSA, the binding of HCV-LPs was detected by flow cytometry using the mouse monoclonal anti-E2 antibody AP33 and PE-conjugated anti-mouse IgG (A and B) or human polyclonal anti-HCV and FITC-conjugated anti-human IgG antibody (C). The fluorescence intensity and relative cell numbers (Counts) are shown on the x and y axes, respectively. NC, negative control, corresponding to HuH-7 cells incubated with control insect cell preparations (GUS) and control serum. (D) For the assessment of concentration-dependent antibody-mediated inhibition of binding, HCV-LPs (genotype 1b) were incubated in subsaturating concentrations with an anti-HCV-positive serum or a pool of anti-HCV-negative control sera (at various dilutions as indicated on the x axis) for 1 h at 37°C. HCV-LP-antibody complexes were added to HuH-7 cells for 1 h at 4°C. HCV-LP binding to the HuH-7 cells was detected by flow cytometry as described above. Inhibition of cellular HCV-LP binding ( y axis) was calculated as described in Materials and Methods.

    Article Snippet: The cells were incubated for 30 min at 4°C with phycoerythrin (PE)-conjugated anti-mouse immunoglobulin G (IgG) antibody (Jackson ImmunoResearch, West Grove, Pa.) or fluorescein isothiocyanate (FITC)-conjugated anti-human IgG antibody (ICN Biomedicals Inc., Aurora, Ohio) diluted in 50 μl of PBS-2% BSA (1:100).

    Techniques: Inhibition, Binding Assay, Incubation, Negative Control, Flow Cytometry, Cytometry, Fluorescence, Concentration Assay

    Th differentiation and B cell responses in mice deprived of functional RLTPR molecules. (A) Sorted naive CD4 + T cells (2 × 10 5 ) from mice of the specified genotype were stimulated for 5 d with anti-CD3 and -CD28 under Th1, Th2, or Th17 differentiating conditions. After 5 d of culture, the absolute number of IFN-γ + (Th1 condition), IL-4 + (Th2 condition), and IL-17 + (Th17 condition) CD4 + T cells was determined. Each dot corresponds to a mouse and the mean (horizontal bar) is indicated. (B) WT and Rltpr bas/bas mice were immunized intraperitoneally at day 0 and 14 with the T cell–dependent antigen TNP-KLH. The concentration of TNP-specific immunoglobulins of the indicated isotypes (IgG2a, IgG2b, and IgG1) were assessed in individual mice before and 21 d after immunization. (C) WT and Rltpr bas/bas mice were immunized with the T cell–independent antigen TNP-LPS, and the concentration of TNP-specific IgM was assessed in individual mice before and 7 d after immunization. (D) Splenic B cells from mice of the specified genotype were stimulated with F(ab)’ 2 goat anti–mouse IgM antibody in the presence or absence of anti-CD40 antibody, or LPS. After 4 d of culture, B cell proliferation was evaluated. Mean and SEM are shown. Data are representative of two independent experiments. In A–C, each dot corresponds to a mouse and the mean (horizontal bar) is indicated. **, P ≤ 0.01; ****, P ≤ 0.001; ns, nonsignificant. In D, two animals were used per genotype.

    Journal: The Journal of Experimental Medicine

    Article Title: The scaffolding function of the RLTPR protein explains its essential role for CD28 co-stimulation in mouse and human T cells

    doi: 10.1084/jem.20160579

    Figure Lengend Snippet: Th differentiation and B cell responses in mice deprived of functional RLTPR molecules. (A) Sorted naive CD4 + T cells (2 × 10 5 ) from mice of the specified genotype were stimulated for 5 d with anti-CD3 and -CD28 under Th1, Th2, or Th17 differentiating conditions. After 5 d of culture, the absolute number of IFN-γ + (Th1 condition), IL-4 + (Th2 condition), and IL-17 + (Th17 condition) CD4 + T cells was determined. Each dot corresponds to a mouse and the mean (horizontal bar) is indicated. (B) WT and Rltpr bas/bas mice were immunized intraperitoneally at day 0 and 14 with the T cell–dependent antigen TNP-KLH. The concentration of TNP-specific immunoglobulins of the indicated isotypes (IgG2a, IgG2b, and IgG1) were assessed in individual mice before and 21 d after immunization. (C) WT and Rltpr bas/bas mice were immunized with the T cell–independent antigen TNP-LPS, and the concentration of TNP-specific IgM was assessed in individual mice before and 7 d after immunization. (D) Splenic B cells from mice of the specified genotype were stimulated with F(ab)’ 2 goat anti–mouse IgM antibody in the presence or absence of anti-CD40 antibody, or LPS. After 4 d of culture, B cell proliferation was evaluated. Mean and SEM are shown. Data are representative of two independent experiments. In A–C, each dot corresponds to a mouse and the mean (horizontal bar) is indicated. **, P ≤ 0.01; ****, P ≤ 0.001; ns, nonsignificant. In D, two animals were used per genotype.

    Article Snippet: CD28 internalization assay Cells were incubated for 30 min on ice with anti-CD28 (1 µg/ml; 553294; BD), followed by incubation for another 30 min on ice with biotinylated goat antibody to hamster immunoglobulin G (2 µg/ml; 107–066-142; Jackson ImmunoResearch Laboratories).

    Techniques: Mouse Assay, Functional Assay, Concentration Assay

    (A,B) Lactoferrin (Lf) fragments accumulate in macrophages following incubation with senescent neutrophils. (A) Peritoneal macrophages were recovered 66 h post zymosan A-induced (1 mg/mouse) peritonitis initiation and incubated with apoptotic Jurkat cells (AC), senescent peritoneal neutrophils (SN), latex beads (LBs), IgG-opsonized LB (oLB), or anti-CD11b monoclonal antibodies as indicated. After 24 h, unbound cells were washed and macrophages were recovered. Then, the cells were lysed, and equal amounts of protein extract were blotted for Lf. Protein extracts from apoptotic neutrophils were also analyzed as indicated. Results are representative from three independent experiments (cells were pooled from three to five mice). (B) Peritoneal neutrophils were recovered 24 h PPI and treated with roscovitine (10 µM) alone or with protease inhibitor cocktail (Pi), cycloheximide (CHX), or the neutrophil elastase inhibitor, silevestat (NEi) for 20 h. Then, neutrophils were lysed, and their protein content was immunoblotted for Lf [results are representative from three experiments (cells were pooled from three to five mice)]. (C,D) Lf fragments are found in interstitial fluids (ISFs) of spleen and inguinal lymph nodes (LN) during peritonitis. Spleen (C) and inguinal LN (D) were harvested 24 and 66 h after initiation of peritonitis ( n = 4–5) and mashed in equal volumes of PBS employing a nylon grid. Equal amounts of ISF protein were run by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), followed by Western blotting for Lf. Results are representative blots from three experiments. (E–G) Peritoneal resolution phase macrophages are essential for the differential production of splenic Lf fragments. Mice were injected intraperitoneally (i.p.) with clodronate-containing or empty liposomes 42 h PPI or before the initiation of peritonitis. After additional 24 h, the spleens were recovered and mashed in equal volumes of PBS employing a nylon grid. Equal amounts of ISF protein were run by 12% SDS-PAGE, followed by Western blotting for Lf. Results are a representative blot (E) and averages of densitometric analysis of the 23- (F) and 17 (G) -kDa bands from three experiments ( n = 3–6 mice per group). **/*indicate statistically significant differences of P ≤ 0.01/ P ≤ 0.05, respectively, by ANOVA with Tukey post hoc analysis. (H) Lf fragments are found in dairy cow milk during the onset and resolution of mastitis. Milk samples from dairy cows with mastitis (days 1–7) were defatted and separated into soluble and cellular fractions by centrifugation. The cellular fraction was lysed and the cytoplasmic proteins were recovered. Equal amounts of protein from both fractions were analyzed by Western blotting for Lf. Results show representative blots from three sample sets taken daily from three different cows.

    Journal: Frontiers in Immunology

    Article Title: A 17-kDa Fragment of Lactoferrin Associates With the Termination of Inflammation and Peptides Within Promote Resolution

    doi: 10.3389/fimmu.2018.00644

    Figure Lengend Snippet: (A,B) Lactoferrin (Lf) fragments accumulate in macrophages following incubation with senescent neutrophils. (A) Peritoneal macrophages were recovered 66 h post zymosan A-induced (1 mg/mouse) peritonitis initiation and incubated with apoptotic Jurkat cells (AC), senescent peritoneal neutrophils (SN), latex beads (LBs), IgG-opsonized LB (oLB), or anti-CD11b monoclonal antibodies as indicated. After 24 h, unbound cells were washed and macrophages were recovered. Then, the cells were lysed, and equal amounts of protein extract were blotted for Lf. Protein extracts from apoptotic neutrophils were also analyzed as indicated. Results are representative from three independent experiments (cells were pooled from three to five mice). (B) Peritoneal neutrophils were recovered 24 h PPI and treated with roscovitine (10 µM) alone or with protease inhibitor cocktail (Pi), cycloheximide (CHX), or the neutrophil elastase inhibitor, silevestat (NEi) for 20 h. Then, neutrophils were lysed, and their protein content was immunoblotted for Lf [results are representative from three experiments (cells were pooled from three to five mice)]. (C,D) Lf fragments are found in interstitial fluids (ISFs) of spleen and inguinal lymph nodes (LN) during peritonitis. Spleen (C) and inguinal LN (D) were harvested 24 and 66 h after initiation of peritonitis ( n = 4–5) and mashed in equal volumes of PBS employing a nylon grid. Equal amounts of ISF protein were run by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), followed by Western blotting for Lf. Results are representative blots from three experiments. (E–G) Peritoneal resolution phase macrophages are essential for the differential production of splenic Lf fragments. Mice were injected intraperitoneally (i.p.) with clodronate-containing or empty liposomes 42 h PPI or before the initiation of peritonitis. After additional 24 h, the spleens were recovered and mashed in equal volumes of PBS employing a nylon grid. Equal amounts of ISF protein were run by 12% SDS-PAGE, followed by Western blotting for Lf. Results are a representative blot (E) and averages of densitometric analysis of the 23- (F) and 17 (G) -kDa bands from three experiments ( n = 3–6 mice per group). **/*indicate statistically significant differences of P ≤ 0.01/ P ≤ 0.05, respectively, by ANOVA with Tukey post hoc analysis. (H) Lf fragments are found in dairy cow milk during the onset and resolution of mastitis. Milk samples from dairy cows with mastitis (days 1–7) were defatted and separated into soluble and cellular fractions by centrifugation. The cellular fraction was lysed and the cytoplasmic proteins were recovered. Equal amounts of protein from both fractions were analyzed by Western blotting for Lf. Results show representative blots from three sample sets taken daily from three different cows.

    Article Snippet: Reagents The following reagents were purchased as detailed: acrylamide/bis-acrylamide, fibronectin, lipopolysaccharide (LPS) (from Escherichia coli , clone 055:B5), staurosporine, TEMED, Tween-20, the neutrophil elastase inhibitor silevestat, and zymosan A from Sigma-Aldrich; clodronate from Liposoma BV; anti-goat horseradish peroxidase-conjugated immunoglobulin G (IgG) and anti-rabbit horseradish peroxidase-conjugated IgG from Jackson Immuno Research Laboratories; WesternBright™ ECL from Advansta, fetal calf serum (FCS), L-glutamine, penicillin–streptomycin, RPMI 1640 and trypan blue from Biological Industries, Kibbutz Beit Haemek; goat anti-mouse CD11b (M-19) polyclonal IgG and rabbit anti-human Lf (H-65) polyclonal IgG from SantaCruz Biotechnology; FITC-conjugated anti-mouse Gr-1, PE-conjugated anti-mouse F4/80, PerCP-conjugated anti-mouse CD11b, APC-conjugated anti-mouse CXCR4 (clone BL6-146508) and enzyme-linked immunosorbent assay (ELISA) kits for TNFα, IL-6, IL-12, and IL-10 from Biolegend; cycloheximide (CHX) from Cayman Chemical; PE selection kit was purchased from Stem Cell Technologies; and Protease Inhibitors Cocktail was purchased from Roche.

    Techniques: Incubation, Mouse Assay, Protease Inhibitor, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Injection, Centrifugation