goat anti mouse igg Millipore Search Results


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  • 90
    Millipore goat anti mouse heavy chain specific igg1
    Effect of co-expressed Sl CYS8 or Sl CDI on C5-1 accumulation. (A) Plants are represented as schematic diagrams where the area of each circle represents the weight of the corresponding leaf. Levels of grey are proportional to the amount of C5-1 on a total soluble protein basis, as determined by quantitative ELISA with a murine <t>IgG</t> standard and normalised to the maximum amount obtained. Leaves were infiltrated with the C5-1 expression vector alone or along with Sl CYS8 or Sl CDI expression vectors. Each value is the mean of three independent (biological) replicates. Numbers below each plant indicate mean antibody production level for the whole plant. (B) Immunodetection of C5-1 at 6 dpi in Leaf 1, Leaf 3, Leaf 5 and Leaf 7, expressed either alone or along with Sl CYS8 or Sl CDI. The proteins were detected with alkaline phosphatase-conjugated goat anti-mouse IgG, after SDS-PAGE under non-reducing conditions. (C) Immunodetection of Sl CYS8 and Sl CDI at 6 dpi in Leaves 1 to 7, following SDS-PAGE under reducing conditions. Sl CYS8 was detected with polyclonal primary antibodies raised in rabbit, Sl CDI with IgY primary antibodies raised in chicken. The primary antibodies were detected with appropriate alkaline phosphatase-conjugated secondary antibodies. EV, mock infiltration with an empty vector.
    Goat Anti Mouse Heavy Chain Specific Igg1, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 5 article reviews
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    94
    Millipore goat anti mouse igg1
    rSjVAMP2-specific IgG, <t>IgG1</t> and IgG2a responses in different groups. Mice were immunized with rSjVAMP2, ISA206 adjuvant, or PBS on weeks 0, 2, and 4, and they were challenged with cercariae on week 6. Serum samples were obtained from ten individual mice from each group on weeks 0, 1, 3, 5, and 12 before sacrifice, and assayed by ELISA. Values depicted are means ± SEM. *** (P
    Goat Anti Mouse Igg1, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 424 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore goat anti mouse igg1 alexa 568
    rSjVAMP2-specific IgG, <t>IgG1</t> and IgG2a responses in different groups. Mice were immunized with rSjVAMP2, ISA206 adjuvant, or PBS on weeks 0, 2, and 4, and they were challenged with cercariae on week 6. Serum samples were obtained from ten individual mice from each group on weeks 0, 1, 3, 5, and 12 before sacrifice, and assayed by ELISA. Values depicted are means ± SEM. *** (P
    Goat Anti Mouse Igg1 Alexa 568, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore goat anti mouse igg1 alexa 488 594
    rSjVAMP2-specific IgG, <t>IgG1</t> and IgG2a responses in different groups. Mice were immunized with rSjVAMP2, ISA206 adjuvant, or PBS on weeks 0, 2, and 4, and they were challenged with cercariae on week 6. Serum samples were obtained from ten individual mice from each group on weeks 0, 1, 3, 5, and 12 before sacrifice, and assayed by ELISA. Values depicted are means ± SEM. *** (P
    Goat Anti Mouse Igg1 Alexa 488 594, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore agarose immobilized goat anti mouse igg1
    Susceptibility of target cells for FasL mediated cell death. ( A ) CEA + LS174T, SW948 and H508 tumor cells and CEA − Jurkat cells for control were incubated in 96-well micro-titer plates in the presence of recombinant CD8 -Fas-L fusion protein (2 µg/mL) for 24 h. The viability of tumor cells was determined by the XTT assay and specific cytolysis calculated. Data represent mean of technical replicates + SD. Significant differences were calculated by the Student’s t test. A representative experiment out of three is shown. ( B ) Resting CD4 + CD25 − T cells express low levels of perforin. CD4 + CD25 − CAR T cells were cultured without stimulation for 48 h and tested by flow cytometry for intracellular perforin levels. An isotype matched mAb served as control for staining. ( C , D ) Perforin and granzyme B were upregulated in CD4 + CD25 − but not in CD4 + CD25 + CAR T cells upon CAR stimulation. CD4 + CD25 − and CD4 + CD25 + CAR T cells (2.5 × 10 4 /well) were stimulated in micro-titer plates coated with 10 µg/mL anti-idiotypic mAb BW2064 or with an isotype control <t>IgG1</t> for 48 h. Cells from triplicates were pooled, stained with anti-perforin ( C ) or anti-granzyme B ( D ) mAbs, and recorded by intracellular flow cytometry.
    Agarose Immobilized Goat Anti Mouse Igg1, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Millipore goat anti mouse igg igg1 and igg2a hrp conjugates
    Serum anti-rTsCB <t>IgG</t> titers determined by rTsCB-ELISA. The OD values are shown as the mean ± SD of anti-rTsCB IgG levels of 20 immunized mice
    Goat Anti Mouse Igg Igg1 And Igg2a Hrp Conjugates, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 81 stars, based on 4 article reviews
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    80
    Millipore fitc conjugated goat anti mouse igg1
    Surface expression of β3 and αV integrin subunits in M. tuberculosis –stimulated monocytes adherent to ECM proteins. Monocytes cultured in the presence or absence of type I collagen or fibronectin were stimulated for 24 h with CoMtb (1:5 dilution), and CoMCont as control, or directly infected with M. tuberculosis (MOI = 1). Cells were fixed, blocked, and stained with <t>FITC-conjugated</t> anti-integrin β1or primary anti-integrin β2, β3, or αV Abs and secondary FITC-conjugated anti-mouse IgG Ab. Secondary Ab alone or FITC-conjugated <t>IgG1</t> isotype Ab were used as controls. ( A ) Histograms of integrin subunits β1, β2, and β3 for control and CoMtb-stimulated monocytes show an increase in β3 expression with CoMtb stimulation. MFIs of integrin subunits ( B ) β3 and ( C ) αV in monocytes stimulated with M. tuberculosis , and MFIs of ( D ) β3 and ( E ) αV in monocytes stimulated with CoMtb, in the presence or absence of matrix components ( n = 4). MFIs were normalized to baseline MFIs of respective controls (control media or CoMCont). (B)–(E) show data points and means. * p
    Fitc Conjugated Goat Anti Mouse Igg1, supplied by Millipore, used in various techniques. Bioz Stars score: 80/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore igg cy2
    Surface expression of β3 and αV integrin subunits in M. tuberculosis –stimulated monocytes adherent to ECM proteins. Monocytes cultured in the presence or absence of type I collagen or fibronectin were stimulated for 24 h with CoMtb (1:5 dilution), and CoMCont as control, or directly infected with M. tuberculosis (MOI = 1). Cells were fixed, blocked, and stained with <t>FITC-conjugated</t> anti-integrin β1or primary anti-integrin β2, β3, or αV Abs and secondary FITC-conjugated anti-mouse IgG Ab. Secondary Ab alone or FITC-conjugated <t>IgG1</t> isotype Ab were used as controls. ( A ) Histograms of integrin subunits β1, β2, and β3 for control and CoMtb-stimulated monocytes show an increase in β3 expression with CoMtb stimulation. MFIs of integrin subunits ( B ) β3 and ( C ) αV in monocytes stimulated with M. tuberculosis , and MFIs of ( D ) β3 and ( E ) αV in monocytes stimulated with CoMtb, in the presence or absence of matrix components ( n = 4). MFIs were normalized to baseline MFIs of respective controls (control media or CoMCont). (B)–(E) show data points and means. * p
    Igg Cy2, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Millipore bengal linked goat anti mice igg
    Surface expression of β3 and αV integrin subunits in M. tuberculosis –stimulated monocytes adherent to ECM proteins. Monocytes cultured in the presence or absence of type I collagen or fibronectin were stimulated for 24 h with CoMtb (1:5 dilution), and CoMCont as control, or directly infected with M. tuberculosis (MOI = 1). Cells were fixed, blocked, and stained with <t>FITC-conjugated</t> anti-integrin β1or primary anti-integrin β2, β3, or αV Abs and secondary FITC-conjugated anti-mouse IgG Ab. Secondary Ab alone or FITC-conjugated <t>IgG1</t> isotype Ab were used as controls. ( A ) Histograms of integrin subunits β1, β2, and β3 for control and CoMtb-stimulated monocytes show an increase in β3 expression with CoMtb stimulation. MFIs of integrin subunits ( B ) β3 and ( C ) αV in monocytes stimulated with M. tuberculosis , and MFIs of ( D ) β3 and ( E ) αV in monocytes stimulated with CoMtb, in the presence or absence of matrix components ( n = 4). MFIs were normalized to baseline MFIs of respective controls (control media or CoMCont). (B)–(E) show data points and means. * p
    Bengal Linked Goat Anti Mice Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA goat anti mouse igg h l
    Surface expression of β3 and αV integrin subunits in M. tuberculosis –stimulated monocytes adherent to ECM proteins. Monocytes cultured in the presence or absence of type I collagen or fibronectin were stimulated for 24 h with CoMtb (1:5 dilution), and CoMCont as control, or directly infected with M. tuberculosis (MOI = 1). Cells were fixed, blocked, and stained with <t>FITC-conjugated</t> anti-integrin β1or primary anti-integrin β2, β3, or αV Abs and secondary FITC-conjugated anti-mouse IgG Ab. Secondary Ab alone or FITC-conjugated <t>IgG1</t> isotype Ab were used as controls. ( A ) Histograms of integrin subunits β1, β2, and β3 for control and CoMtb-stimulated monocytes show an increase in β3 expression with CoMtb stimulation. MFIs of integrin subunits ( B ) β3 and ( C ) αV in monocytes stimulated with M. tuberculosis , and MFIs of ( D ) β3 and ( E ) αV in monocytes stimulated with CoMtb, in the presence or absence of matrix components ( n = 4). MFIs were normalized to baseline MFIs of respective controls (control media or CoMCont). (B)–(E) show data points and means. * p
    Goat Anti Mouse Igg H L, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore goat anti mouse igg cross linker
    Surface expression of β3 and αV integrin subunits in M. tuberculosis –stimulated monocytes adherent to ECM proteins. Monocytes cultured in the presence or absence of type I collagen or fibronectin were stimulated for 24 h with CoMtb (1:5 dilution), and CoMCont as control, or directly infected with M. tuberculosis (MOI = 1). Cells were fixed, blocked, and stained with <t>FITC-conjugated</t> anti-integrin β1or primary anti-integrin β2, β3, or αV Abs and secondary FITC-conjugated anti-mouse IgG Ab. Secondary Ab alone or FITC-conjugated <t>IgG1</t> isotype Ab were used as controls. ( A ) Histograms of integrin subunits β1, β2, and β3 for control and CoMtb-stimulated monocytes show an increase in β3 expression with CoMtb stimulation. MFIs of integrin subunits ( B ) β3 and ( C ) αV in monocytes stimulated with M. tuberculosis , and MFIs of ( D ) β3 and ( E ) αV in monocytes stimulated with CoMtb, in the presence or absence of matrix components ( n = 4). MFIs were normalized to baseline MFIs of respective controls (control media or CoMCont). (B)–(E) show data points and means. * p
    Goat Anti Mouse Igg Cross Linker, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore alexa goat anti mouse igg antibodies
    Surface expression of β3 and αV integrin subunits in M. tuberculosis –stimulated monocytes adherent to ECM proteins. Monocytes cultured in the presence or absence of type I collagen or fibronectin were stimulated for 24 h with CoMtb (1:5 dilution), and CoMCont as control, or directly infected with M. tuberculosis (MOI = 1). Cells were fixed, blocked, and stained with <t>FITC-conjugated</t> anti-integrin β1or primary anti-integrin β2, β3, or αV Abs and secondary FITC-conjugated anti-mouse IgG Ab. Secondary Ab alone or FITC-conjugated <t>IgG1</t> isotype Ab were used as controls. ( A ) Histograms of integrin subunits β1, β2, and β3 for control and CoMtb-stimulated monocytes show an increase in β3 expression with CoMtb stimulation. MFIs of integrin subunits ( B ) β3 and ( C ) αV in monocytes stimulated with M. tuberculosis , and MFIs of ( D ) β3 and ( E ) αV in monocytes stimulated with CoMtb, in the presence or absence of matrix components ( n = 4). MFIs were normalized to baseline MFIs of respective controls (control media or CoMCont). (B)–(E) show data points and means. * p
    Alexa Goat Anti Mouse Igg Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore goat anti mouse igg phycoerythrin conjugate
    Surface expression of β3 and αV integrin subunits in M. tuberculosis –stimulated monocytes adherent to ECM proteins. Monocytes cultured in the presence or absence of type I collagen or fibronectin were stimulated for 24 h with CoMtb (1:5 dilution), and CoMCont as control, or directly infected with M. tuberculosis (MOI = 1). Cells were fixed, blocked, and stained with <t>FITC-conjugated</t> anti-integrin β1or primary anti-integrin β2, β3, or αV Abs and secondary FITC-conjugated anti-mouse IgG Ab. Secondary Ab alone or FITC-conjugated <t>IgG1</t> isotype Ab were used as controls. ( A ) Histograms of integrin subunits β1, β2, and β3 for control and CoMtb-stimulated monocytes show an increase in β3 expression with CoMtb stimulation. MFIs of integrin subunits ( B ) β3 and ( C ) αV in monocytes stimulated with M. tuberculosis , and MFIs of ( D ) β3 and ( E ) αV in monocytes stimulated with CoMtb, in the presence or absence of matrix components ( n = 4). MFIs were normalized to baseline MFIs of respective controls (control media or CoMCont). (B)–(E) show data points and means. * p
    Goat Anti Mouse Igg Phycoerythrin Conjugate, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore polyvalent goat anti mouse igg antibody
    Surface expression of β3 and αV integrin subunits in M. tuberculosis –stimulated monocytes adherent to ECM proteins. Monocytes cultured in the presence or absence of type I collagen or fibronectin were stimulated for 24 h with CoMtb (1:5 dilution), and CoMCont as control, or directly infected with M. tuberculosis (MOI = 1). Cells were fixed, blocked, and stained with <t>FITC-conjugated</t> anti-integrin β1or primary anti-integrin β2, β3, or αV Abs and secondary FITC-conjugated anti-mouse IgG Ab. Secondary Ab alone or FITC-conjugated <t>IgG1</t> isotype Ab were used as controls. ( A ) Histograms of integrin subunits β1, β2, and β3 for control and CoMtb-stimulated monocytes show an increase in β3 expression with CoMtb stimulation. MFIs of integrin subunits ( B ) β3 and ( C ) αV in monocytes stimulated with M. tuberculosis , and MFIs of ( D ) β3 and ( E ) αV in monocytes stimulated with CoMtb, in the presence or absence of matrix components ( n = 4). MFIs were normalized to baseline MFIs of respective controls (control media or CoMCont). (B)–(E) show data points and means. * p
    Polyvalent Goat Anti Mouse Igg Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore goat anti mice cy3 conjugated igg
    Surface expression of β3 and αV integrin subunits in M. tuberculosis –stimulated monocytes adherent to ECM proteins. Monocytes cultured in the presence or absence of type I collagen or fibronectin were stimulated for 24 h with CoMtb (1:5 dilution), and CoMCont as control, or directly infected with M. tuberculosis (MOI = 1). Cells were fixed, blocked, and stained with <t>FITC-conjugated</t> anti-integrin β1or primary anti-integrin β2, β3, or αV Abs and secondary FITC-conjugated anti-mouse IgG Ab. Secondary Ab alone or FITC-conjugated <t>IgG1</t> isotype Ab were used as controls. ( A ) Histograms of integrin subunits β1, β2, and β3 for control and CoMtb-stimulated monocytes show an increase in β3 expression with CoMtb stimulation. MFIs of integrin subunits ( B ) β3 and ( C ) αV in monocytes stimulated with M. tuberculosis , and MFIs of ( D ) β3 and ( E ) αV in monocytes stimulated with CoMtb, in the presence or absence of matrix components ( n = 4). MFIs were normalized to baseline MFIs of respective controls (control media or CoMCont). (B)–(E) show data points and means. * p
    Goat Anti Mice Cy3 Conjugated Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Millipore goat anti mouse igg gold antibody
    Surface expression of β3 and αV integrin subunits in M. tuberculosis –stimulated monocytes adherent to ECM proteins. Monocytes cultured in the presence or absence of type I collagen or fibronectin were stimulated for 24 h with CoMtb (1:5 dilution), and CoMCont as control, or directly infected with M. tuberculosis (MOI = 1). Cells were fixed, blocked, and stained with <t>FITC-conjugated</t> anti-integrin β1or primary anti-integrin β2, β3, or αV Abs and secondary FITC-conjugated anti-mouse IgG Ab. Secondary Ab alone or FITC-conjugated <t>IgG1</t> isotype Ab were used as controls. ( A ) Histograms of integrin subunits β1, β2, and β3 for control and CoMtb-stimulated monocytes show an increase in β3 expression with CoMtb stimulation. MFIs of integrin subunits ( B ) β3 and ( C ) αV in monocytes stimulated with M. tuberculosis , and MFIs of ( D ) β3 and ( E ) αV in monocytes stimulated with CoMtb, in the presence or absence of matrix components ( n = 4). MFIs were normalized to baseline MFIs of respective controls (control media or CoMCont). (B)–(E) show data points and means. * p
    Goat Anti Mouse Igg Gold Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 82/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore goat anti mouse fitc conjugate igg
    Surface expression of β3 and αV integrin subunits in M. tuberculosis –stimulated monocytes adherent to ECM proteins. Monocytes cultured in the presence or absence of type I collagen or fibronectin were stimulated for 24 h with CoMtb (1:5 dilution), and CoMCont as control, or directly infected with M. tuberculosis (MOI = 1). Cells were fixed, blocked, and stained with <t>FITC-conjugated</t> anti-integrin β1or primary anti-integrin β2, β3, or αV Abs and secondary FITC-conjugated anti-mouse IgG Ab. Secondary Ab alone or FITC-conjugated <t>IgG1</t> isotype Ab were used as controls. ( A ) Histograms of integrin subunits β1, β2, and β3 for control and CoMtb-stimulated monocytes show an increase in β3 expression with CoMtb stimulation. MFIs of integrin subunits ( B ) β3 and ( C ) αV in monocytes stimulated with M. tuberculosis , and MFIs of ( D ) β3 and ( E ) αV in monocytes stimulated with CoMtb, in the presence or absence of matrix components ( n = 4). MFIs were normalized to baseline MFIs of respective controls (control media or CoMCont). (B)–(E) show data points and means. * p
    Goat Anti Mouse Fitc Conjugate Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore biotinylated goat anti mouse igg conjugate
    Surface expression of β3 and αV integrin subunits in M. tuberculosis –stimulated monocytes adherent to ECM proteins. Monocytes cultured in the presence or absence of type I collagen or fibronectin were stimulated for 24 h with CoMtb (1:5 dilution), and CoMCont as control, or directly infected with M. tuberculosis (MOI = 1). Cells were fixed, blocked, and stained with <t>FITC-conjugated</t> anti-integrin β1or primary anti-integrin β2, β3, or αV Abs and secondary FITC-conjugated anti-mouse IgG Ab. Secondary Ab alone or FITC-conjugated <t>IgG1</t> isotype Ab were used as controls. ( A ) Histograms of integrin subunits β1, β2, and β3 for control and CoMtb-stimulated monocytes show an increase in β3 expression with CoMtb stimulation. MFIs of integrin subunits ( B ) β3 and ( C ) αV in monocytes stimulated with M. tuberculosis , and MFIs of ( D ) β3 and ( E ) αV in monocytes stimulated with CoMtb, in the presence or absence of matrix components ( n = 4). MFIs were normalized to baseline MFIs of respective controls (control media or CoMCont). (B)–(E) show data points and means. * p
    Biotinylated Goat Anti Mouse Igg Conjugate, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore goat anti mouse igg alkaline phosphatase
    Specific antibody response in mice immunized with untargeted (SAG1t) or targeted (SA2) proteins. Detection of specific anti-SAG1 <t>IgG</t> antibodies (A) and IgG subclasses (B) in sera of mice (12/group) primed and boosted twice with SAG1t, SA2, or phosphate-buffered saline formulated with Poly I:C by combined routes. Serum samples collected after the last boost were tested by ELISA using SAG1t protein as the coating antigen. Results are expressed as the mean ± SEM ( n = 12) of log2 titers and represent one of two independent experiments. *** P
    Goat Anti Mouse Igg Alkaline Phosphatase, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore goat anti mouse igg crosslinking antibody sigma
    Specific antibody response in mice immunized with untargeted (SAG1t) or targeted (SA2) proteins. Detection of specific anti-SAG1 <t>IgG</t> antibodies (A) and IgG subclasses (B) in sera of mice (12/group) primed and boosted twice with SAG1t, SA2, or phosphate-buffered saline formulated with Poly I:C by combined routes. Serum samples collected after the last boost were tested by ELISA using SAG1t protein as the coating antigen. Results are expressed as the mean ± SEM ( n = 12) of log2 titers and represent one of two independent experiments. *** P
    Goat Anti Mouse Igg Crosslinking Antibody Sigma, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Millipore gold conjugated goat anti mouse igg
    Specific antibody response in mice immunized with untargeted (SAG1t) or targeted (SA2) proteins. Detection of specific anti-SAG1 <t>IgG</t> antibodies (A) and IgG subclasses (B) in sera of mice (12/group) primed and boosted twice with SAG1t, SA2, or phosphate-buffered saline formulated with Poly I:C by combined routes. Serum samples collected after the last boost were tested by ELISA using SAG1t protein as the coating antigen. Results are expressed as the mean ± SEM ( n = 12) of log2 titers and represent one of two independent experiments. *** P
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    Specific antibody response in mice immunized with untargeted (SAG1t) or targeted (SA2) proteins. Detection of specific anti-SAG1 <t>IgG</t> antibodies (A) and IgG subclasses (B) in sera of mice (12/group) primed and boosted twice with SAG1t, SA2, or phosphate-buffered saline formulated with Poly I:C by combined routes. Serum samples collected after the last boost were tested by ELISA using SAG1t protein as the coating antigen. Results are expressed as the mean ± SEM ( n = 12) of log2 titers and represent one of two independent experiments. *** P
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    Specific antibody response in mice immunized with untargeted (SAG1t) or targeted (SA2) proteins. Detection of specific anti-SAG1 <t>IgG</t> antibodies (A) and IgG subclasses (B) in sera of mice (12/group) primed and boosted twice with SAG1t, SA2, or phosphate-buffered saline formulated with Poly I:C by combined routes. Serum samples collected after the last boost were tested by ELISA using SAG1t protein as the coating antigen. Results are expressed as the mean ± SEM ( n = 12) of log2 titers and represent one of two independent experiments. *** P
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    Specific antibody response in mice immunized with untargeted (SAG1t) or targeted (SA2) proteins. Detection of specific anti-SAG1 <t>IgG</t> antibodies (A) and IgG subclasses (B) in sera of mice (12/group) primed and boosted twice with SAG1t, SA2, or phosphate-buffered saline formulated with Poly I:C by combined routes. Serum samples collected after the last boost were tested by ELISA using SAG1t protein as the coating antigen. Results are expressed as the mean ± SEM ( n = 12) of log2 titers and represent one of two independent experiments. *** P
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    Specific antibody response in mice immunized with untargeted (SAG1t) or targeted (SA2) proteins. Detection of specific anti-SAG1 <t>IgG</t> antibodies (A) and IgG subclasses (B) in sera of mice (12/group) primed and boosted twice with SAG1t, SA2, or phosphate-buffered saline formulated with Poly I:C by combined routes. Serum samples collected after the last boost were tested by ELISA using SAG1t protein as the coating antigen. Results are expressed as the mean ± SEM ( n = 12) of log2 titers and represent one of two independent experiments. *** P
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    Specific antibody response in mice immunized with untargeted (SAG1t) or targeted (SA2) proteins. Detection of specific anti-SAG1 <t>IgG</t> antibodies (A) and IgG subclasses (B) in sera of mice (12/group) primed and boosted twice with SAG1t, SA2, or phosphate-buffered saline formulated with Poly I:C by combined routes. Serum samples collected after the last boost were tested by ELISA using SAG1t protein as the coating antigen. Results are expressed as the mean ± SEM ( n = 12) of log2 titers and represent one of two independent experiments. *** P
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    Specific antibody response in mice immunized with untargeted (SAG1t) or targeted (SA2) proteins. Detection of specific anti-SAG1 <t>IgG</t> antibodies (A) and IgG subclasses (B) in sera of mice (12/group) primed and boosted twice with SAG1t, SA2, or phosphate-buffered saline formulated with Poly I:C by combined routes. Serum samples collected after the last boost were tested by ELISA using SAG1t protein as the coating antigen. Results are expressed as the mean ± SEM ( n = 12) of log2 titers and represent one of two independent experiments. *** P
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    Specific antibody response in mice immunized with untargeted (SAG1t) or targeted (SA2) proteins. Detection of specific anti-SAG1 <t>IgG</t> antibodies (A) and IgG subclasses (B) in sera of mice (12/group) primed and boosted twice with SAG1t, SA2, or phosphate-buffered saline formulated with Poly I:C by combined routes. Serum samples collected after the last boost were tested by ELISA using SAG1t protein as the coating antigen. Results are expressed as the mean ± SEM ( n = 12) of log2 titers and represent one of two independent experiments. *** P
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    Specific antibody response in mice immunized with untargeted (SAG1t) or targeted (SA2) proteins. Detection of specific anti-SAG1 <t>IgG</t> antibodies (A) and IgG subclasses (B) in sera of mice (12/group) primed and boosted twice with SAG1t, SA2, or phosphate-buffered saline formulated with Poly I:C by combined routes. Serum samples collected after the last boost were tested by ELISA using SAG1t protein as the coating antigen. Results are expressed as the mean ± SEM ( n = 12) of log2 titers and represent one of two independent experiments. *** P
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    Specific antibody response in mice immunized with untargeted (SAG1t) or targeted (SA2) proteins. Detection of specific anti-SAG1 <t>IgG</t> antibodies (A) and IgG subclasses (B) in sera of mice (12/group) primed and boosted twice with SAG1t, SA2, or phosphate-buffered saline formulated with Poly I:C by combined routes. Serum samples collected after the last boost were tested by ELISA using SAG1t protein as the coating antigen. Results are expressed as the mean ± SEM ( n = 12) of log2 titers and represent one of two independent experiments. *** P
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    Image Search Results


    Effect of co-expressed Sl CYS8 or Sl CDI on C5-1 accumulation. (A) Plants are represented as schematic diagrams where the area of each circle represents the weight of the corresponding leaf. Levels of grey are proportional to the amount of C5-1 on a total soluble protein basis, as determined by quantitative ELISA with a murine IgG standard and normalised to the maximum amount obtained. Leaves were infiltrated with the C5-1 expression vector alone or along with Sl CYS8 or Sl CDI expression vectors. Each value is the mean of three independent (biological) replicates. Numbers below each plant indicate mean antibody production level for the whole plant. (B) Immunodetection of C5-1 at 6 dpi in Leaf 1, Leaf 3, Leaf 5 and Leaf 7, expressed either alone or along with Sl CYS8 or Sl CDI. The proteins were detected with alkaline phosphatase-conjugated goat anti-mouse IgG, after SDS-PAGE under non-reducing conditions. (C) Immunodetection of Sl CYS8 and Sl CDI at 6 dpi in Leaves 1 to 7, following SDS-PAGE under reducing conditions. Sl CYS8 was detected with polyclonal primary antibodies raised in rabbit, Sl CDI with IgY primary antibodies raised in chicken. The primary antibodies were detected with appropriate alkaline phosphatase-conjugated secondary antibodies. EV, mock infiltration with an empty vector.

    Journal: PLoS ONE

    Article Title: Protection of Recombinant Mammalian Antibodies from Development-Dependent Proteolysis in Leaves of Nicotiana benthamiana

    doi: 10.1371/journal.pone.0070203

    Figure Lengend Snippet: Effect of co-expressed Sl CYS8 or Sl CDI on C5-1 accumulation. (A) Plants are represented as schematic diagrams where the area of each circle represents the weight of the corresponding leaf. Levels of grey are proportional to the amount of C5-1 on a total soluble protein basis, as determined by quantitative ELISA with a murine IgG standard and normalised to the maximum amount obtained. Leaves were infiltrated with the C5-1 expression vector alone or along with Sl CYS8 or Sl CDI expression vectors. Each value is the mean of three independent (biological) replicates. Numbers below each plant indicate mean antibody production level for the whole plant. (B) Immunodetection of C5-1 at 6 dpi in Leaf 1, Leaf 3, Leaf 5 and Leaf 7, expressed either alone or along with Sl CYS8 or Sl CDI. The proteins were detected with alkaline phosphatase-conjugated goat anti-mouse IgG, after SDS-PAGE under non-reducing conditions. (C) Immunodetection of Sl CYS8 and Sl CDI at 6 dpi in Leaves 1 to 7, following SDS-PAGE under reducing conditions. Sl CYS8 was detected with polyclonal primary antibodies raised in rabbit, Sl CDI with IgY primary antibodies raised in chicken. The primary antibodies were detected with appropriate alkaline phosphatase-conjugated secondary antibodies. EV, mock infiltration with an empty vector.

    Article Snippet: C5-1 Quantification Enzyme-linked immunosorbent assay (ELISA) plates for C5-1 quantification (Becton Dickinson, Mississauga ON, Canada) were coated with 3.75 µg/mL goat anti-mouse heavy chain-specific IgG1 (Sigma-Aldrich) in 50 mM carbonate buffer (pH 9.0) at 4°C for 16–18 h. The plates were washed three times in 10 mM phosphate-buffered saline containing 0.1% (v/v) Tween 20 (PBS-T), blocked through a 1-h incubation in 1% (w/v) casein in phosphate-buffered saline (PBS) (Pierce, Rockford IL, USA) at 37°C, and washed three times in PBS-T. A standard curve was generated for each plate with 0, 4, 8, 16, 24, 32, 40 and 60 ng/mL of purified mouse IgG1 (Sigma-Aldrich).

    Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Plasmid Preparation, Immunodetection, SDS Page

    Biological activity of C5-1 expressed alone or along with Sl CYS8 or Sl CDI. (A) Plants are represented as schematic diagrams, where the area of each circle represents the weight of the corresponding leaf. Levels of grey represent relative C5-1 activities (absorbance units) on a total soluble protein basis, as measured by ELISA and normalised to the maximum amount obtained. Each value is the mean of three biological replicates. (B) Immunodetection of C5-1 activity at 6 dpi in Leaf 1, Leaf 3, Leaf 5 and Leaf 7, with the recombinant antibody expressed alone or along with Sl CYS8 or Sl CDI. Antibody activity was inferred from binding of human IgG following electroblotting of non-reduced SDS-PAGE-separated proteins onto nitrocellulose sheets. The human IgG antigen of C5-1 was then detected with a goat anti-human conjugated with alkaline phosphatase. EV, mock infiltration with an empty vector.

    Journal: PLoS ONE

    Article Title: Protection of Recombinant Mammalian Antibodies from Development-Dependent Proteolysis in Leaves of Nicotiana benthamiana

    doi: 10.1371/journal.pone.0070203

    Figure Lengend Snippet: Biological activity of C5-1 expressed alone or along with Sl CYS8 or Sl CDI. (A) Plants are represented as schematic diagrams, where the area of each circle represents the weight of the corresponding leaf. Levels of grey represent relative C5-1 activities (absorbance units) on a total soluble protein basis, as measured by ELISA and normalised to the maximum amount obtained. Each value is the mean of three biological replicates. (B) Immunodetection of C5-1 activity at 6 dpi in Leaf 1, Leaf 3, Leaf 5 and Leaf 7, with the recombinant antibody expressed alone or along with Sl CYS8 or Sl CDI. Antibody activity was inferred from binding of human IgG following electroblotting of non-reduced SDS-PAGE-separated proteins onto nitrocellulose sheets. The human IgG antigen of C5-1 was then detected with a goat anti-human conjugated with alkaline phosphatase. EV, mock infiltration with an empty vector.

    Article Snippet: C5-1 Quantification Enzyme-linked immunosorbent assay (ELISA) plates for C5-1 quantification (Becton Dickinson, Mississauga ON, Canada) were coated with 3.75 µg/mL goat anti-mouse heavy chain-specific IgG1 (Sigma-Aldrich) in 50 mM carbonate buffer (pH 9.0) at 4°C for 16–18 h. The plates were washed three times in 10 mM phosphate-buffered saline containing 0.1% (v/v) Tween 20 (PBS-T), blocked through a 1-h incubation in 1% (w/v) casein in phosphate-buffered saline (PBS) (Pierce, Rockford IL, USA) at 37°C, and washed three times in PBS-T. A standard curve was generated for each plate with 0, 4, 8, 16, 24, 32, 40 and 60 ng/mL of purified mouse IgG1 (Sigma-Aldrich).

    Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Immunodetection, Recombinant, Binding Assay, SDS Page, Plasmid Preparation

    rSjVAMP2-specific IgG, IgG1 and IgG2a responses in different groups. Mice were immunized with rSjVAMP2, ISA206 adjuvant, or PBS on weeks 0, 2, and 4, and they were challenged with cercariae on week 6. Serum samples were obtained from ten individual mice from each group on weeks 0, 1, 3, 5, and 12 before sacrifice, and assayed by ELISA. Values depicted are means ± SEM. *** (P

    Journal: PLoS ONE

    Article Title: Characterization of VAMP2 in Schistosoma japonicum and the Evaluation of Protective Efficacy Induced by Recombinant SjVAMP2 in Mice

    doi: 10.1371/journal.pone.0144584

    Figure Lengend Snippet: rSjVAMP2-specific IgG, IgG1 and IgG2a responses in different groups. Mice were immunized with rSjVAMP2, ISA206 adjuvant, or PBS on weeks 0, 2, and 4, and they were challenged with cercariae on week 6. Serum samples were obtained from ten individual mice from each group on weeks 0, 1, 3, 5, and 12 before sacrifice, and assayed by ELISA. Values depicted are means ± SEM. *** (P

    Article Snippet: To detect IgG1 and IgG2a, goat anti-mouse IgG1- or IgG2a-conjugated HRP (Sigma–Aldrich) (1:2000 dilutions) were used as secondary antibodies as described for IgG detection.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Specific total IgG responses in experimental groups after first (Fig 2a.), second (Fig 2b.) and third (Fig 2c.) immunization rounds. Immunization with both vaccines raised total IgG after the first and second immunization, with no significant difference between them. Following the third immunization, HBsAg formulated in Montanide ISA 266 showed superiority in the induction of total IgG versus the commercial vaccine

    Journal: Gastroenterology and Hepatology From Bed to Bench

    Article Title: Formulation of HBs antigen in Montanide ISA266 shows superiority to commercial HBsAg vaccine in the induction of humoral immune responses

    doi:

    Figure Lengend Snippet: Specific total IgG responses in experimental groups after first (Fig 2a.), second (Fig 2b.) and third (Fig 2c.) immunization rounds. Immunization with both vaccines raised total IgG after the first and second immunization, with no significant difference between them. Following the third immunization, HBsAg formulated in Montanide ISA 266 showed superiority in the induction of total IgG versus the commercial vaccine

    Article Snippet: To detect specific IgG1, IgG2a, goat anti-mouse IgG1, IgG2a secondary antibodies (Sigma, USA) were used according to the manufacturer’s instruction.

    Techniques:

    Specific IgG1 (Fig 3a.) and IgG2a (Fig 3b.) antibodies in the experimental groups. Results of particular IgG1/IgG2a shows a higher response in the group that immunized with HBsAg formulated in Montanide ISA 266 versus the commercial vaccine

    Journal: Gastroenterology and Hepatology From Bed to Bench

    Article Title: Formulation of HBs antigen in Montanide ISA266 shows superiority to commercial HBsAg vaccine in the induction of humoral immune responses

    doi:

    Figure Lengend Snippet: Specific IgG1 (Fig 3a.) and IgG2a (Fig 3b.) antibodies in the experimental groups. Results of particular IgG1/IgG2a shows a higher response in the group that immunized with HBsAg formulated in Montanide ISA 266 versus the commercial vaccine

    Article Snippet: To detect specific IgG1, IgG2a, goat anti-mouse IgG1, IgG2a secondary antibodies (Sigma, USA) were used according to the manufacturer’s instruction.

    Techniques:

    Immunization with rSsEno with or without PGA conjugation enhanced the antibody response. BALB/c mice were s.c. immunized three times with rSsEno100, PGA + rSsEno100 or PBS as a negative control. Sera collected seven days after the last boost was used to determine antigen-specific IgG ( A ), IgG1 ( B ), IgG2a ( C ), and IgG3 ( D ) titers by ELISA. The results are presented as the mean ± SD of 5 mice from one of three independent experiments, and statistical significance was determined by one-way ANOVA using Tukey’s multiple comparisons test and a 95% confidence interval. *(p

    Journal: Scientific Reports

    Article Title: Immunization with recombinant enolase of Sporothrix spp. (rSsEno) confers effective protection against sporotrichosis in mice

    doi: 10.1038/s41598-019-53135-z

    Figure Lengend Snippet: Immunization with rSsEno with or without PGA conjugation enhanced the antibody response. BALB/c mice were s.c. immunized three times with rSsEno100, PGA + rSsEno100 or PBS as a negative control. Sera collected seven days after the last boost was used to determine antigen-specific IgG ( A ), IgG1 ( B ), IgG2a ( C ), and IgG3 ( D ) titers by ELISA. The results are presented as the mean ± SD of 5 mice from one of three independent experiments, and statistical significance was determined by one-way ANOVA using Tukey’s multiple comparisons test and a 95% confidence interval. *(p

    Article Snippet: After three washes with PBS, the strips were further incubated for 2 h with goat anti-mouse IgG (Sigma-Aldrich) diluted 1:500 or goat anti-feline IgG (Southern Biotech) diluted 1:1000.

    Techniques: Conjugation Assay, Mouse Assay, Negative Control, Enzyme-linked Immunosorbent Assay

    Anti-TeNT antibody titre in the plasma and anti-TeNT antibody-forming cells in the spleen. (a) Titres of anti-TeNT IgM, IgG1 and IgG2a in plasma were evaluated by ELISA. Results are expressed as mean of log 2 titre ± SE of control mice (open bars) and of RST mice (filled bars). (b) Anti-TeNT-specific AFCs IgM and IgG were quantified by ELISPOT. Results are expressed as mean ± SE of control mice (open bars) and of RST mice (filled bars). Anti-TeNT IgG and anti-TeNT IgG FACs were undetectable on day 5. * P

    Journal: Immunology

    Article Title: Chronic restraint stress induces severe disruption of the T-cell specific response to tetanus toxin vaccine

    doi: 10.1046/j.1365-2567.2001.01152.x

    Figure Lengend Snippet: Anti-TeNT antibody titre in the plasma and anti-TeNT antibody-forming cells in the spleen. (a) Titres of anti-TeNT IgM, IgG1 and IgG2a in plasma were evaluated by ELISA. Results are expressed as mean of log 2 titre ± SE of control mice (open bars) and of RST mice (filled bars). (b) Anti-TeNT-specific AFCs IgM and IgG were quantified by ELISPOT. Results are expressed as mean ± SE of control mice (open bars) and of RST mice (filled bars). Anti-TeNT IgG and anti-TeNT IgG FACs were undetectable on day 5. * P

    Article Snippet: Ninety-six-well nitrocellulose plates (MAHA millipore, Bedford, MA) were coated overnight at room temperature with goat IgG anti-mouse IgG (Sigma) or IgM (Bethyl, Montgomery, TX) at 5 µg/ml (100 µl per well) for total IgG or IgM AFCs, respectively, or with TeNT at 5 µg/ml for anti-TeNT-specific AFCs.

    Techniques: Enzyme-linked Immunosorbent Assay, Mouse Assay, Enzyme-linked Immunospot, FACS

    Blockade of Sema4a but not IFNαR1 signaling increases bronchiolitis severity and predisposes to subsequent asthma. (a) Protocol of PVM inoculation and administration of αSema4a or isotype control (IgG2a) and αIFNαR or isotype control (IgG1) to neonatal WT mice. (b) IFN-α protein expression in BALF, expressed as % change of the respective isotype control. (c) Viral load in the airway epithelium. (d) Nrp-1 + T reg cells in the lung. (e) IL-10 protein expression in lung, expressed as % change of the respective isotype control. (f) CD39 expression by Nrp-1 + T reg cells. (g) Sloughing of the airway epithelium. (h) Eosinophil number in BAL. (i) IL-33 protein expression in lung, expressed as % change of the respective isotype control. (j) Type-2 ILCs in lung. (k) AHR. (l) ASM area. (m) Intraluminal mucus plugging. (n) Eosinophil number in BAL. (o) IL-13 protein expression in BAL. Data are representative of n = 2 experiments with four to six neonates in each group and are presented as the mean ± SEM (k) or box-and-whisker plots showing quartiles (boxes) and range (whiskers; b–o). Data were analyzed by one-way (b–j and l–o) or two-way (k) ANOVA with Tukey’s post hoc test; *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Plasmacytoid dendritic cells protect from viral bronchiolitis and asthma through semaphorin 4a–mediated T reg expansion

    doi: 10.1084/jem.20170298

    Figure Lengend Snippet: Blockade of Sema4a but not IFNαR1 signaling increases bronchiolitis severity and predisposes to subsequent asthma. (a) Protocol of PVM inoculation and administration of αSema4a or isotype control (IgG2a) and αIFNαR or isotype control (IgG1) to neonatal WT mice. (b) IFN-α protein expression in BALF, expressed as % change of the respective isotype control. (c) Viral load in the airway epithelium. (d) Nrp-1 + T reg cells in the lung. (e) IL-10 protein expression in lung, expressed as % change of the respective isotype control. (f) CD39 expression by Nrp-1 + T reg cells. (g) Sloughing of the airway epithelium. (h) Eosinophil number in BAL. (i) IL-33 protein expression in lung, expressed as % change of the respective isotype control. (j) Type-2 ILCs in lung. (k) AHR. (l) ASM area. (m) Intraluminal mucus plugging. (n) Eosinophil number in BAL. (o) IL-13 protein expression in BAL. Data are representative of n = 2 experiments with four to six neonates in each group and are presented as the mean ± SEM (k) or box-and-whisker plots showing quartiles (boxes) and range (whiskers; b–o). Data were analyzed by one-way (b–j and l–o) or two-way (k) ANOVA with Tukey’s post hoc test; *, P

    Article Snippet: Sections were washed three times in PBS/0.05% Tween-20 before incubation with biotinylated anti-mouse polyclonal antibody (Invitrogen; for PVM detection), goat anti–rabbit IgG-AP (Sigma-Aldrich; periostin), or goat anti–mouse IgG-AP (Sigma-Aldrich; α-smooth muscle actin).

    Techniques: Mouse Assay, Expressing, Whisker Assay

    Down-regulation of surface MHC class I expression by γHV-68 K3 and its KSHV homologs. The γHV-68 K3, the KSHV K3, and the KSHV K5 ORFs were cloned into pMSCV-GFP and expressed by retroviral transduction, as indicated in human 293 cells stably transfected with murine H-2K b and murine L929 cells stably transfected with human HLA-A2. GFP + transduced cells were then mixed with GFP − untransduced cells to compare MHC class I expression, in each case using a phycoerythrin-conjugated goat anti-mouse IgG secondary antibody.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Inhibition of MHC class I-restricted antigen presentation by ?2-herpesviruses

    doi:

    Figure Lengend Snippet: Down-regulation of surface MHC class I expression by γHV-68 K3 and its KSHV homologs. The γHV-68 K3, the KSHV K3, and the KSHV K5 ORFs were cloned into pMSCV-GFP and expressed by retroviral transduction, as indicated in human 293 cells stably transfected with murine H-2K b and murine L929 cells stably transfected with human HLA-A2. GFP + transduced cells were then mixed with GFP − untransduced cells to compare MHC class I expression, in each case using a phycoerythrin-conjugated goat anti-mouse IgG secondary antibody.

    Article Snippet: Cells were washed in ice-cold PBS/BSA (0.1%)/azide (0.01%) and stained for H-2Kb (Y3), H-2Db (28.14.8), H-2Kd (SF-1.1.1), or HLA-ABC (W6/32), followed by rabbit anti-mouse-IgG-fluorescein isothiocyanate (Dako) or goat anti-mouse-IgG-phycoerythrin (Sigma–Aldrich).

    Techniques: Expressing, Clone Assay, Transduction, Stable Transfection, Transfection

    Intranasal immunisation with lipoproteins induces lower local and systemic humoral immune responses. (A) Six C57BL/6 mice used for the in vivo colonisation model were randomly selected for the analysis of their antibody kinetics following an intranasal immunisation with the lipoproteins MetQ, DacB, and PnrA. The mice received three doses with 5 μg antigen and 4 μg CTB as the adjuvant in 2-week intervals. Before each treatment and 2 weeks after the third immunisation, antisera were collected to determine the antibody kinetics. The data from each individual mouse are depicted for every protein. Antisera were serially diluted and measured using the FLEXMAP 3D® system. The response values reflect the levels of antigen-specific IgG. (B,C) Three weeks after the final immunisation, the mice were challenged with S. pneumoniae D39 (3.4 × 10 6 CFU) and 3 days after infection their nasal tissues were harvested, homogenised and analysed for local antigen-specific IgG (B) and IgA (C) using ELISA. The IgG and IgA levels were determined using a 1:10 or 1:2 dilution of the nasal homogenate, respectively. The data were statistically analysed using a Mann-Whitney U -test. Symbols represent individual mice ( n = 12) and the bars represent the group median. * p

    Journal: Frontiers in Immunology

    Article Title: Intranasal Vaccination With Lipoproteins Confers Protection Against Pneumococcal Colonisation

    doi: 10.3389/fimmu.2018.02405

    Figure Lengend Snippet: Intranasal immunisation with lipoproteins induces lower local and systemic humoral immune responses. (A) Six C57BL/6 mice used for the in vivo colonisation model were randomly selected for the analysis of their antibody kinetics following an intranasal immunisation with the lipoproteins MetQ, DacB, and PnrA. The mice received three doses with 5 μg antigen and 4 μg CTB as the adjuvant in 2-week intervals. Before each treatment and 2 weeks after the third immunisation, antisera were collected to determine the antibody kinetics. The data from each individual mouse are depicted for every protein. Antisera were serially diluted and measured using the FLEXMAP 3D® system. The response values reflect the levels of antigen-specific IgG. (B,C) Three weeks after the final immunisation, the mice were challenged with S. pneumoniae D39 (3.4 × 10 6 CFU) and 3 days after infection their nasal tissues were harvested, homogenised and analysed for local antigen-specific IgG (B) and IgA (C) using ELISA. The IgG and IgA levels were determined using a 1:10 or 1:2 dilution of the nasal homogenate, respectively. The data were statistically analysed using a Mann-Whitney U -test. Symbols represent individual mice ( n = 12) and the bars represent the group median. * p

    Article Snippet: The plates were washed and incubated for 1 h at RT with horseradish peroxidase coupled with rabbit anti-mouse total IgG (Jackson ImmunoResearch Laboratories, Inc.), rabbit anti-mouse IgG1 (Sigma-Aldrich), goat anti-mouse IgG2a (Sigma-Aldrich), goat anti-mouse IgG2c (Abcam, Cambridge, UK), or goat anti-mouse IgA antibody (Sigma-Aldrich).

    Techniques: Mouse Assay, In Vivo, CtB Assay, Infection, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    Pneumococcal lipoproteins are highly abundant on the pneumococcal surface. (A) In immunoblots, the specificity of antisera derived from intraperitoneal immunisations of CD-1 mice ( n = 6) with recombinant lipoproteins was assessed. Therefore, the wild-type strain S. pneumoniae D39Δ cps and the corresponding isogenic lipoprotein deficient mutants (2 × 10 8 bacteria per lane) were used. Enolase was detected with a rabbit anti-enolase serum and served as a loading control. (B) IgG antibody titrations were performed by incubating equimolar amounts of recombinant proteins with serial dilutions of isolated polyclonal IgGs. Detection was carried out using a peroxidase-coupled goat anti-mouse IgG followed by incubation with OPD as a substrate and absorbance was measured at 492 nm. Titrations were performed at least three times and the error bars represent the SEM. (C,D) Using the equation for the hyperbolic regression curve ( y [ A b s ] = B m a x · x K d + x , Bmax, maximal binding; Kd, concentration for half maximal binding) an initial IgG concentration was calculated in the linear dynamic range. The polyclonal IgGs with equal contents of IgG specific for each lipoprotein were therefore applied to enable the comparison of their surface abundances. In a flow cytometric approach, D39Δ cps (C) and D39 (D) were incubated with the appropriate calculated concentration of IgG and concentrations 5-, 10-, 20-, and 50-fold greater to analyse the surface abundance of the selected lipoproteins. Antibody binding was detected using a goat anti-mouse Alexa Fluor® 488-coupled secondary antibody. The percentage of positive gated events is depicted in the graphs, thereby indicating the proportion of wild-type bacteria positive for the binding of the respective anti-lipoprotein IgGs. The mean values of at least three independent experiments are shown, with error bars corresponding to SEM.

    Journal: Frontiers in Immunology

    Article Title: Intranasal Vaccination With Lipoproteins Confers Protection Against Pneumococcal Colonisation

    doi: 10.3389/fimmu.2018.02405

    Figure Lengend Snippet: Pneumococcal lipoproteins are highly abundant on the pneumococcal surface. (A) In immunoblots, the specificity of antisera derived from intraperitoneal immunisations of CD-1 mice ( n = 6) with recombinant lipoproteins was assessed. Therefore, the wild-type strain S. pneumoniae D39Δ cps and the corresponding isogenic lipoprotein deficient mutants (2 × 10 8 bacteria per lane) were used. Enolase was detected with a rabbit anti-enolase serum and served as a loading control. (B) IgG antibody titrations were performed by incubating equimolar amounts of recombinant proteins with serial dilutions of isolated polyclonal IgGs. Detection was carried out using a peroxidase-coupled goat anti-mouse IgG followed by incubation with OPD as a substrate and absorbance was measured at 492 nm. Titrations were performed at least three times and the error bars represent the SEM. (C,D) Using the equation for the hyperbolic regression curve ( y [ A b s ] = B m a x · x K d + x , Bmax, maximal binding; Kd, concentration for half maximal binding) an initial IgG concentration was calculated in the linear dynamic range. The polyclonal IgGs with equal contents of IgG specific for each lipoprotein were therefore applied to enable the comparison of their surface abundances. In a flow cytometric approach, D39Δ cps (C) and D39 (D) were incubated with the appropriate calculated concentration of IgG and concentrations 5-, 10-, 20-, and 50-fold greater to analyse the surface abundance of the selected lipoproteins. Antibody binding was detected using a goat anti-mouse Alexa Fluor® 488-coupled secondary antibody. The percentage of positive gated events is depicted in the graphs, thereby indicating the proportion of wild-type bacteria positive for the binding of the respective anti-lipoprotein IgGs. The mean values of at least three independent experiments are shown, with error bars corresponding to SEM.

    Article Snippet: The plates were washed and incubated for 1 h at RT with horseradish peroxidase coupled with rabbit anti-mouse total IgG (Jackson ImmunoResearch Laboratories, Inc.), rabbit anti-mouse IgG1 (Sigma-Aldrich), goat anti-mouse IgG2a (Sigma-Aldrich), goat anti-mouse IgG2c (Abcam, Cambridge, UK), or goat anti-mouse IgA antibody (Sigma-Aldrich).

    Techniques: Western Blot, Derivative Assay, Mouse Assay, Recombinant, Isolation, Incubation, Binding Assay, Concentration Assay, Flow Cytometry

    Analysis of convalescent patient sera and mouse sera derived from intraperitoneal immunisations indicate the high immunogenicity of PnrA, DacB, and MetQ. (A) A total of 22 antisera from convalescent patients who suffered from pneumococcal infections such as pneumonia ( n = 6), meningitis ( n = 7), sepsis ( n = 4), and unknown clinical outcomes ( n = 5) caused by different pneumococcal serotypes were analysed to compare their levels of anti-lipoprotein antibodies. Each symbol represents a single antiserum, while the different colours indicate the clinical outcome of every patient. (B) The immunogenicity of the lipoproteins was further demonstrated by analysing the antibody kinetics of intraperitoneally immunised CD-1 mice ( n = 6). The mice received three vaccinations with 20 μg antigen and Alum as the adjuvant, with a 2-week interval between treatments. Before each treatment and 2 weeks after the final immunisation, antisera were collected to enable the determination of the antibody kinetics. Each individual mouse is depicted in the graphs. All serum samples were serially diluted and measured using the FLEXMAP 3D® system. Response values reflect the levels of antigen-specific IgG for the 11 tested lipoproteins and the positive control PspA.

    Journal: Frontiers in Immunology

    Article Title: Intranasal Vaccination With Lipoproteins Confers Protection Against Pneumococcal Colonisation

    doi: 10.3389/fimmu.2018.02405

    Figure Lengend Snippet: Analysis of convalescent patient sera and mouse sera derived from intraperitoneal immunisations indicate the high immunogenicity of PnrA, DacB, and MetQ. (A) A total of 22 antisera from convalescent patients who suffered from pneumococcal infections such as pneumonia ( n = 6), meningitis ( n = 7), sepsis ( n = 4), and unknown clinical outcomes ( n = 5) caused by different pneumococcal serotypes were analysed to compare their levels of anti-lipoprotein antibodies. Each symbol represents a single antiserum, while the different colours indicate the clinical outcome of every patient. (B) The immunogenicity of the lipoproteins was further demonstrated by analysing the antibody kinetics of intraperitoneally immunised CD-1 mice ( n = 6). The mice received three vaccinations with 20 μg antigen and Alum as the adjuvant, with a 2-week interval between treatments. Before each treatment and 2 weeks after the final immunisation, antisera were collected to enable the determination of the antibody kinetics. Each individual mouse is depicted in the graphs. All serum samples were serially diluted and measured using the FLEXMAP 3D® system. Response values reflect the levels of antigen-specific IgG for the 11 tested lipoproteins and the positive control PspA.

    Article Snippet: The plates were washed and incubated for 1 h at RT with horseradish peroxidase coupled with rabbit anti-mouse total IgG (Jackson ImmunoResearch Laboratories, Inc.), rabbit anti-mouse IgG1 (Sigma-Aldrich), goat anti-mouse IgG2a (Sigma-Aldrich), goat anti-mouse IgG2c (Abcam, Cambridge, UK), or goat anti-mouse IgA antibody (Sigma-Aldrich).

    Techniques: Derivative Assay, Mouse Assay, Positive Control

    Intraperitoneal and intranasal immunisations with DacB, MetQ, or PnrA predominantly induce IgG1 responses. Antigen-specific total IgG, IgG1, and IgG2 titres were monitored using an ELISA in either post-immune sera following an intraperitoneal immunisation with Alum as the adjuvant (A,B) or in post-challenge sera obtained after intranasal immunisation with CTB as the adjuvant followed by an intranasal challenge with S. pneumoniae D39 (C,D) . Antibody titres of each serum specimen are denoted as the log 10 of the reciprocal dilution of the serum giving twice the average absorbance of the sera derived from the PBS-treated group. The data were statistically analysed using a Mann-Whitney U -test. Symbols represent individual mice ( n = 6 for Alum group, n = 12 for CTB group) and bars represent the group median. * p

    Journal: Frontiers in Immunology

    Article Title: Intranasal Vaccination With Lipoproteins Confers Protection Against Pneumococcal Colonisation

    doi: 10.3389/fimmu.2018.02405

    Figure Lengend Snippet: Intraperitoneal and intranasal immunisations with DacB, MetQ, or PnrA predominantly induce IgG1 responses. Antigen-specific total IgG, IgG1, and IgG2 titres were monitored using an ELISA in either post-immune sera following an intraperitoneal immunisation with Alum as the adjuvant (A,B) or in post-challenge sera obtained after intranasal immunisation with CTB as the adjuvant followed by an intranasal challenge with S. pneumoniae D39 (C,D) . Antibody titres of each serum specimen are denoted as the log 10 of the reciprocal dilution of the serum giving twice the average absorbance of the sera derived from the PBS-treated group. The data were statistically analysed using a Mann-Whitney U -test. Symbols represent individual mice ( n = 6 for Alum group, n = 12 for CTB group) and bars represent the group median. * p

    Article Snippet: The plates were washed and incubated for 1 h at RT with horseradish peroxidase coupled with rabbit anti-mouse total IgG (Jackson ImmunoResearch Laboratories, Inc.), rabbit anti-mouse IgG1 (Sigma-Aldrich), goat anti-mouse IgG2a (Sigma-Aldrich), goat anti-mouse IgG2c (Abcam, Cambridge, UK), or goat anti-mouse IgA antibody (Sigma-Aldrich).

    Techniques: Enzyme-linked Immunosorbent Assay, CtB Assay, Derivative Assay, MANN-WHITNEY, Mouse Assay

    Pneumococcal lipoproteins heterologously expressed in E. coli . A 1-μg aliquot of heterologously expressed pneumococcal lipoproteins was separated using SDS-PAGE and the proteins were detected using silver staining (A) or with immunoblotting using a monoclonal mouse anti-Penta-His 6 antibody and alkaline phosphatase-conjugated goat anti-mouse IgG (B) .

    Journal: Frontiers in Immunology

    Article Title: Intranasal Vaccination With Lipoproteins Confers Protection Against Pneumococcal Colonisation

    doi: 10.3389/fimmu.2018.02405

    Figure Lengend Snippet: Pneumococcal lipoproteins heterologously expressed in E. coli . A 1-μg aliquot of heterologously expressed pneumococcal lipoproteins was separated using SDS-PAGE and the proteins were detected using silver staining (A) or with immunoblotting using a monoclonal mouse anti-Penta-His 6 antibody and alkaline phosphatase-conjugated goat anti-mouse IgG (B) .

    Article Snippet: The plates were washed and incubated for 1 h at RT with horseradish peroxidase coupled with rabbit anti-mouse total IgG (Jackson ImmunoResearch Laboratories, Inc.), rabbit anti-mouse IgG1 (Sigma-Aldrich), goat anti-mouse IgG2a (Sigma-Aldrich), goat anti-mouse IgG2c (Abcam, Cambridge, UK), or goat anti-mouse IgA antibody (Sigma-Aldrich).

    Techniques: SDS Page, Silver Staining

    The gp42 linker mutants are expressed independently of gHgL expression and are not functional in B cell fusion. CHO-KI cells were transiently transfected with 2 µg of the gp42 linker mutants either in the presence or absence of gHgL and gB (0.5 µg each) and T7 luciferase (0.8 µg). Eighteen hours posttransfection, 40,000 cells were transferred to each well of a black 96-well plate and overlaid with 40,000 Daudi B cells for fusion assay. Eighteen hours after overlay, cells were washed with phosphate-buffered saline (PBS) and lysed for 10 min with 50 µl of passive lysis buffer (Promega) per well. Luciferase activity was measured with a PerkinElmer Victor plate reader immediately after addition of 50 µl/well of luciferase reagent (Promega). Eighteen hours posttransfection, 80,000 cells were transferred to each well of a clear 96-well plate, and eighteen hours later, surface expression was determined by cELISA using anti-gp42 (3H3), fixation, secondary biotinylated anti-mouse IgG antibody (Sigma), tertiary streptavidin-horseradish peroxidase (GE Healthcare) and TMB (3,3=,5,5=-tetramethylbenzidine) after TMB one-component substrate (BioFX). Absorbance readings were taken at 380 nm using a PerkinElmer Victor plate reader. Binding was normalized to wild-type gp42 binding levels, which were set at 100%. Data shown are representative of the results from three independent experiments.

    Journal: mBio

    Article Title: Membrane Anchoring of Epstein-Barr Virus gp42 Inhibits Fusion with B Cells Even with Increased Flexibility Allowed by Engineered Spacers

    doi: 10.1128/mBio.02285-14

    Figure Lengend Snippet: The gp42 linker mutants are expressed independently of gHgL expression and are not functional in B cell fusion. CHO-KI cells were transiently transfected with 2 µg of the gp42 linker mutants either in the presence or absence of gHgL and gB (0.5 µg each) and T7 luciferase (0.8 µg). Eighteen hours posttransfection, 40,000 cells were transferred to each well of a black 96-well plate and overlaid with 40,000 Daudi B cells for fusion assay. Eighteen hours after overlay, cells were washed with phosphate-buffered saline (PBS) and lysed for 10 min with 50 µl of passive lysis buffer (Promega) per well. Luciferase activity was measured with a PerkinElmer Victor plate reader immediately after addition of 50 µl/well of luciferase reagent (Promega). Eighteen hours posttransfection, 80,000 cells were transferred to each well of a clear 96-well plate, and eighteen hours later, surface expression was determined by cELISA using anti-gp42 (3H3), fixation, secondary biotinylated anti-mouse IgG antibody (Sigma), tertiary streptavidin-horseradish peroxidase (GE Healthcare) and TMB (3,3=,5,5=-tetramethylbenzidine) after TMB one-component substrate (BioFX). Absorbance readings were taken at 380 nm using a PerkinElmer Victor plate reader. Binding was normalized to wild-type gp42 binding levels, which were set at 100%. Data shown are representative of the results from three independent experiments.

    Article Snippet: The cells were washed, fixed, and incubated with biotinylated goat anti-mouse IgG (Sigma) for 30 min at room temperature, followed by incubation with streptavidin-horseradish peroxidase (HRP) (GE Healthcare) for 30 min at room temperature and with 3,3′,5,5′-tetramethylbenzidine (TMB) one-component HRP substrate (BioFX).

    Techniques: Expressing, Functional Assay, Transfection, Luciferase, Single Vesicle Fusion Assay, Lysis, Activity Assay, Binding Assay

    The gp42 linker mutants bind exogenous gHgL and HLA class II comparable to wild-type gp42. CHO-K1 cells were transiently transfected with each of the d37-41 gp42 linker mutants. Twenty-four hours later, the cells were overlaid with sDQ2-αII (HLA class II) purified protein or sgHgL supernatants (isolated 48 h posttransfection). Protein was overlaid for 1 h at 4°C, and unbound protein was washed away. Binding of HLA class II and sgHgL was determined by cELISA using anti-HLA class II DQ antibody (1a3) (catalog no. ab24265; Abcam) and anti-FLAG-M2 (catalog no. F1804; Sigma), secondary biotinylated anti-mouse IgG antibody, tertiary streptavidin-HRP and TMB substrate and compared to expression using anti-gp42 antibody (3H3). Absorbance readings were taken at 380 nm using a PerkinElmer Victor plate reader. Binding was normalized to wild-type gp42 binding levels, which were set at 100%. Data shown are the average values of three independent experiments.

    Journal: mBio

    Article Title: Membrane Anchoring of Epstein-Barr Virus gp42 Inhibits Fusion with B Cells Even with Increased Flexibility Allowed by Engineered Spacers

    doi: 10.1128/mBio.02285-14

    Figure Lengend Snippet: The gp42 linker mutants bind exogenous gHgL and HLA class II comparable to wild-type gp42. CHO-K1 cells were transiently transfected with each of the d37-41 gp42 linker mutants. Twenty-four hours later, the cells were overlaid with sDQ2-αII (HLA class II) purified protein or sgHgL supernatants (isolated 48 h posttransfection). Protein was overlaid for 1 h at 4°C, and unbound protein was washed away. Binding of HLA class II and sgHgL was determined by cELISA using anti-HLA class II DQ antibody (1a3) (catalog no. ab24265; Abcam) and anti-FLAG-M2 (catalog no. F1804; Sigma), secondary biotinylated anti-mouse IgG antibody, tertiary streptavidin-HRP and TMB substrate and compared to expression using anti-gp42 antibody (3H3). Absorbance readings were taken at 380 nm using a PerkinElmer Victor plate reader. Binding was normalized to wild-type gp42 binding levels, which were set at 100%. Data shown are the average values of three independent experiments.

    Article Snippet: The cells were washed, fixed, and incubated with biotinylated goat anti-mouse IgG (Sigma) for 30 min at room temperature, followed by incubation with streptavidin-horseradish peroxidase (HRP) (GE Healthcare) for 30 min at room temperature and with 3,3′,5,5′-tetramethylbenzidine (TMB) one-component HRP substrate (BioFX).

    Techniques: Transfection, Purification, Isolation, Binding Assay, Expressing

    Kidney pathology in autoimmune pTg18R mice. A, kidney sections stained with goat anti-mouse IgG-FITC at a low magnification (10x). B, IgG and complement factor 3 (C3) deposition revealed by immunofluorescence, and glomerular inflammation revealed by H E

    Journal: Lupus

    Article Title: Spontaneous Autoimmunity in Mice That Carry an IghV Partial Transgene: A Required Arginine in VHCDR3

    doi: 10.1177/0961203308097480

    Figure Lengend Snippet: Kidney pathology in autoimmune pTg18R mice. A, kidney sections stained with goat anti-mouse IgG-FITC at a low magnification (10x). B, IgG and complement factor 3 (C3) deposition revealed by immunofluorescence, and glomerular inflammation revealed by H E

    Article Snippet: HEp-2 staining was performed on NOVA-Lite HEp-2 cells (Inova Diagnostics, San Diego, CA) using sera at a dilution of 1:50 followed by FITC goat anti-mouse IgG (heavy chain-specific) (Sigma, St. Louis, MO).

    Techniques: Mouse Assay, Staining, Immunofluorescence

    Membrane anchoring of MD-2 via TLR4. A stable line expressing MD-2 with the flag epitope was stained with the anti-flag mAb, followed by goat anti–mouse IgG–FITC (a and d). Staining with cell permeabilization is shown in d. Another stable transfectant was used that expressed MD-2 with the protein C epitope and TLR4 in b, c, and e. Cell surface MD-2 and TLR4 are shown with the rat anti–protein C mAb or the mouse anti–human TLR4 mAb HTA125 (b and c, respectively). MD-2 expression in permeabilized cells is shown in e. The second reagents used were goat anti–rat IgG–PE or goat anti–mouse IgG–FITC, respectively. Control histograms stained with isotype-matched mouse or rat mAb are shown (dotted lines).

    Journal: The Journal of Experimental Medicine

    Article Title: MD-2, a Molecule that Confers Lipopolysaccharide Responsiveness on Toll-like Receptor 4

    doi:

    Figure Lengend Snippet: Membrane anchoring of MD-2 via TLR4. A stable line expressing MD-2 with the flag epitope was stained with the anti-flag mAb, followed by goat anti–mouse IgG–FITC (a and d). Staining with cell permeabilization is shown in d. Another stable transfectant was used that expressed MD-2 with the protein C epitope and TLR4 in b, c, and e. Cell surface MD-2 and TLR4 are shown with the rat anti–protein C mAb or the mouse anti–human TLR4 mAb HTA125 (b and c, respectively). MD-2 expression in permeabilized cells is shown in e. The second reagents used were goat anti–rat IgG–PE or goat anti–mouse IgG–FITC, respectively. Control histograms stained with isotype-matched mouse or rat mAb are shown (dotted lines).

    Article Snippet: After washes with staining buffer (PBS containing 2% FCS and 0.1% azide), goat anti–mouse IgG–FITC (Chemicon International, Inc.) or goat anti–rat IgG–PE (Southern Biotechnology Associates, Inc.) was added.

    Techniques: Expressing, FLAG-tag, Staining, Transfection

    MD-2 is coprecipitated with TLR4. (A) Stable transfectants expressing MD-2 alone or with TLR4 were stained with or without cell permeabilization. The intracellular MD-2 precursor was stained with the anti-protein C mAb. The specificity of the anti-TLR4 mAb was shown by cell surface staining of stable transfectants. Goat anti– mouse IgG–FITC was used as the second reagent. Dotted lines correspond to histograms stained with the second reagent alone. Panels correspond to the original Ba/F3 line, a transfectant expressing TLR4 alone, and a transfectant expressing TLR4 + MD-2, respectively. (B) Stable transfectants expressing TLR4 and MD-2 were subjected to immunoprecipitation with control mouse IgG (lane 1) or the anti-TLR4 mAb, HTA125 (lanes 2 and 3). After blotting, precipitated molecules were detected with an anti-flag mAb, M2. The precipitates shown in lanes 1 and 2 are from the transfectant expressing TLR4 and MD-2, both of which were tagged with the flag epitope. In lane 3, we used the control line in which the flag epitope was on TLR4 but not on MD-2.

    Journal: The Journal of Experimental Medicine

    Article Title: MD-2, a Molecule that Confers Lipopolysaccharide Responsiveness on Toll-like Receptor 4

    doi:

    Figure Lengend Snippet: MD-2 is coprecipitated with TLR4. (A) Stable transfectants expressing MD-2 alone or with TLR4 were stained with or without cell permeabilization. The intracellular MD-2 precursor was stained with the anti-protein C mAb. The specificity of the anti-TLR4 mAb was shown by cell surface staining of stable transfectants. Goat anti– mouse IgG–FITC was used as the second reagent. Dotted lines correspond to histograms stained with the second reagent alone. Panels correspond to the original Ba/F3 line, a transfectant expressing TLR4 alone, and a transfectant expressing TLR4 + MD-2, respectively. (B) Stable transfectants expressing TLR4 and MD-2 were subjected to immunoprecipitation with control mouse IgG (lane 1) or the anti-TLR4 mAb, HTA125 (lanes 2 and 3). After blotting, precipitated molecules were detected with an anti-flag mAb, M2. The precipitates shown in lanes 1 and 2 are from the transfectant expressing TLR4 and MD-2, both of which were tagged with the flag epitope. In lane 3, we used the control line in which the flag epitope was on TLR4 but not on MD-2.

    Article Snippet: After washes with staining buffer (PBS containing 2% FCS and 0.1% azide), goat anti–mouse IgG–FITC (Chemicon International, Inc.) or goat anti–rat IgG–PE (Southern Biotechnology Associates, Inc.) was added.

    Techniques: Expressing, Staining, Transfection, Immunoprecipitation, FLAG-tag

    Co-localization of SARS-CoV NP and p42 in 2BS cells . ( A ) SARS-CoV NP was expressed in 2BS cells transfected with recombinant -pcDNA3.0+SNP22b (left panel), 2BS cells transfected with pcDNA3.0 (right panel; negative control). ( B ) Proteasome subunit p42 in 2BS cells localized with anti-p42 antibody and anti-rabbit IgG-FITC conjugate, excitation at 488 nm (lane 1). SARS-CoV NP expressed in 2BS cells, localized using an anti-SARS-CoV NP mAb and anti-mouse IgG-TRITC conjugate, excitation at 568 nm (lane 2). Co-localization was detected by merging lanes 1 and 2 (lane 3).

    Journal: Virology Journal

    Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42

    doi: 10.1186/1743-422X-7-99

    Figure Lengend Snippet: Co-localization of SARS-CoV NP and p42 in 2BS cells . ( A ) SARS-CoV NP was expressed in 2BS cells transfected with recombinant -pcDNA3.0+SNP22b (left panel), 2BS cells transfected with pcDNA3.0 (right panel; negative control). ( B ) Proteasome subunit p42 in 2BS cells localized with anti-p42 antibody and anti-rabbit IgG-FITC conjugate, excitation at 488 nm (lane 1). SARS-CoV NP expressed in 2BS cells, localized using an anti-SARS-CoV NP mAb and anti-mouse IgG-TRITC conjugate, excitation at 568 nm (lane 2). Co-localization was detected by merging lanes 1 and 2 (lane 3).

    Article Snippet: After incubation with a 1:80 dilution of goat anti-rabbit IgG-FITC (Sigma) and a 1:200 dilution of goat anti-mouse IgG-TRITC (Sigma) conjugates at 37°C for 45 min, coverslips were again washed six times in distilled water and air-dried before being mounted on a slide with interspaces containing 50% (v/v) glycerol.

    Techniques: Transfection, Recombinant, Negative Control

    Active β-catenin and cleaved caspase-3 are co-localized in FSH-primed but not LH-stimulated granulosa cells (GCs). Cells were double immunostained with anti-active β-catenin (red) and anti-active casapse-3 (green). DNA was observed by using DAPI (blue). A–C) Cytoplasmic immunostaining of active β-catenin and active caspase-3 in untreated (control) GCs. D–F) Nuclear localization of active β-catenin and active caspase-3 in FSH-primed GCs. G–I) Cytoplasmic immunostaining of active β-catenin and active caspase-3 in LH-stimulated cells. J–K) The negative control was performed by using IgG1 mouse isotypic control as primary antibody. Images are at 200× magnification.

    Journal: Avicenna Journal of Medical Biotechnology

    Article Title: Exogenous Secreted Frizzled-Related Protein-4 Modulates Steroidogenesis of Rat Granulosa Cells through Wnt/β-catenin and PI3K/AKT Signaling Pathways

    doi:

    Figure Lengend Snippet: Active β-catenin and cleaved caspase-3 are co-localized in FSH-primed but not LH-stimulated granulosa cells (GCs). Cells were double immunostained with anti-active β-catenin (red) and anti-active casapse-3 (green). DNA was observed by using DAPI (blue). A–C) Cytoplasmic immunostaining of active β-catenin and active caspase-3 in untreated (control) GCs. D–F) Nuclear localization of active β-catenin and active caspase-3 in FSH-primed GCs. G–I) Cytoplasmic immunostaining of active β-catenin and active caspase-3 in LH-stimulated cells. J–K) The negative control was performed by using IgG1 mouse isotypic control as primary antibody. Images are at 200× magnification.

    Article Snippet: Mouse monoclonal antiactive β-catenin (ABC) which is specific against nuclear β-catenin, and goat anti-mouse IgG-rhodamine were obtained from Millipore (Massachusetts, USA), whereas rabbit polyclonal anti-cleaved caspase-3 antibody was from Calbiochem (Massachusetts, USA), EMD Chemicals, goat anti-rabbit IgG-FITC, rabbit anti-goat IgG-HRP conjugated and IgG1 mouse isotypic control were from Vector Laboratories (Peterborough, UK).

    Techniques: Immunostaining, Negative Control

    BTBR and BCF1 mice have significantly higher amounts of serum anti-brain antibodies than CBF1 or C57BL/6J (B6) mice. Sera from postnatal day 21 mice were added to wells coated with BALB/c SCID whole brain proteins (10 µg/well). The numbers of sera were 6 for BTBR male or female, 6 for BCF1 male, 3 for BCF1 female, 5 for CBF1 male or female, and 7 for B6 male or female. The level of brain-reactive IgG was determined by using the HRP-conjugated goat anti-mouse IgG. *, p

    Journal: PLoS ONE

    Article Title: Aberrant Immune Responses in a Mouse with Behavioral Disorders

    doi: 10.1371/journal.pone.0020912

    Figure Lengend Snippet: BTBR and BCF1 mice have significantly higher amounts of serum anti-brain antibodies than CBF1 or C57BL/6J (B6) mice. Sera from postnatal day 21 mice were added to wells coated with BALB/c SCID whole brain proteins (10 µg/well). The numbers of sera were 6 for BTBR male or female, 6 for BCF1 male, 3 for BCF1 female, 5 for CBF1 male or female, and 7 for B6 male or female. The level of brain-reactive IgG was determined by using the HRP-conjugated goat anti-mouse IgG. *, p

    Article Snippet: IgG ELISA The level of total IgG in serum or brain homogenates was determined by a sandwich ELISA using goat anti-mouse IgG Fc (Pierce, Rockford, IL) as a capture Ab and HRP-goat anti-mouse IgG as a detection Ab (Sigma).

    Techniques: Mouse Assay

    The Δ sdbA mutant does not secrete Sth1 bacteriocins. Secreted bacteriocins were isolated from culture supernatants using Sth 1 -specific rabbit IgG-protein A-Sepharose beads and were detected with a mouse anti-Sth 1 antibody in an enzyme-linked immunosorbent assay (ELISA). (A) Detection of Sth 1 captured from 25 ml of culture supernatant prepared from the parent strain, the Δ sdbA mutant, the sdbA -complemented mutant (SdbA Compl), or uninoculated BHI medium with 5% serum (BHIS) (negative control). (B) Detection of Sth 1 captured from 25 or 100 ml of culture supernatant from the parent strain, the Δ sdbA mutant, or the Δ sdbA mutant induced with exogenous CSP for 30 min. Results are means ± SDs from three experiments. ***, P

    Journal: Journal of Bacteriology

    Article Title: Mutation of the Thiol-Disulfide Oxidoreductase SdbA Activates the CiaRH Two-Component System, Leading to Bacteriocin Expression Shutdown in Streptococcus gordonii

    doi: 10.1128/JB.00800-15

    Figure Lengend Snippet: The Δ sdbA mutant does not secrete Sth1 bacteriocins. Secreted bacteriocins were isolated from culture supernatants using Sth 1 -specific rabbit IgG-protein A-Sepharose beads and were detected with a mouse anti-Sth 1 antibody in an enzyme-linked immunosorbent assay (ELISA). (A) Detection of Sth 1 captured from 25 ml of culture supernatant prepared from the parent strain, the Δ sdbA mutant, the sdbA -complemented mutant (SdbA Compl), or uninoculated BHI medium with 5% serum (BHIS) (negative control). (B) Detection of Sth 1 captured from 25 or 100 ml of culture supernatant from the parent strain, the Δ sdbA mutant, or the Δ sdbA mutant induced with exogenous CSP for 30 min. Results are means ± SDs from three experiments. ***, P

    Article Snippet: Sth1 was detected using goat anti-mouse IgG–biotin (1:20,000; Sigma-Aldrich), followed by ExtrAvidin-alkaline phosphatase (1:60,000; Sigma-Aldrich).

    Techniques: Mutagenesis, Isolation, Enzyme-linked Immunosorbent Assay, Negative Control

    Bac HCVpp associate with human hepatocyte cells. Human HepaRG hepatocytes incubated with bac HCVpp for two hours are shown (left). Binding assay was performed using primary anti-E1 (H4) monoclonal antibody and visualized with Cy3 -labeled secondary anti-IgG antibody. Negative controls are also shown (right). Negative control 1, primary antibody was omitted; negative control 2, bac Flupp was used instead of bac HCVpp. DIC, Differential Interference Contrast

    Journal: Virology Journal

    Article Title: New insights into HCV replication in original cells from Aedes mosquitoes

    doi: 10.1186/s12985-017-0828-z

    Figure Lengend Snippet: Bac HCVpp associate with human hepatocyte cells. Human HepaRG hepatocytes incubated with bac HCVpp for two hours are shown (left). Binding assay was performed using primary anti-E1 (H4) monoclonal antibody and visualized with Cy3 -labeled secondary anti-IgG antibody. Negative controls are also shown (right). Negative control 1, primary antibody was omitted; negative control 2, bac Flupp was used instead of bac HCVpp. DIC, Differential Interference Contrast

    Article Snippet: The anti-E1 mouse IgGs which reacted with bac HCVpp bound to cells were then detected by incubation for one hour with Cy3-coupled goat anti-mouse IgG (Sigma–Aldrich), at a dilution of 1/1000 using a motorized inverted Olympus IE81 microscope and differential interference contrast (DIC).

    Techniques: BAC Assay, Incubation, Binding Assay, Labeling, Negative Control

    Bac HCVpp associate with Aedes mosquito cell lines C6/36 and Aag-2. Mosquito cells incubated with bac HCVpp for two hours are shown (left). Binding assay was performed using primary anti-E1 (H4) monoclonal antibody and visualized with Cy3 -labeled secondary anti-IgG antibody. Negative controls are also shown (right). Negative control 1, primary antibody was omitted; negative control 2, bac Flupp was used instead of bac HCVpp. DIC, Differential Interference Contrast

    Journal: Virology Journal

    Article Title: New insights into HCV replication in original cells from Aedes mosquitoes

    doi: 10.1186/s12985-017-0828-z

    Figure Lengend Snippet: Bac HCVpp associate with Aedes mosquito cell lines C6/36 and Aag-2. Mosquito cells incubated with bac HCVpp for two hours are shown (left). Binding assay was performed using primary anti-E1 (H4) monoclonal antibody and visualized with Cy3 -labeled secondary anti-IgG antibody. Negative controls are also shown (right). Negative control 1, primary antibody was omitted; negative control 2, bac Flupp was used instead of bac HCVpp. DIC, Differential Interference Contrast

    Article Snippet: The anti-E1 mouse IgGs which reacted with bac HCVpp bound to cells were then detected by incubation for one hour with Cy3-coupled goat anti-mouse IgG (Sigma–Aldrich), at a dilution of 1/1000 using a motorized inverted Olympus IE81 microscope and differential interference contrast (DIC).

    Techniques: BAC Assay, Incubation, Binding Assay, Labeling, Negative Control

    OX40L deficiency ameliorates the phenotype of B6. Sle16 lupus-prone mice. Comparison between female B6. Sle16 and B6.Sle16.Tnfsf4 −/− female mice at 9 months of age. (A) Quantitation of spleen/body weight ratio and spleen weight. (B) Absolute number of cells per spleen. (C) Serum level of IgG and IgM at 6 and 9 months. (D) Titre of IgM anti-dsDNA and anti-ssDNA. (E) Quantitation of naïve (T N ) (CD4+, CD62L+, CD44 low ), (T EFF ) effector (CD4+, CD62L low , CD44 low ), T EM effector/memory (CD4+, CD62L low/neg , CD44 hi ) and T CM central/memory (CD4+, CD62L+, CD44 hi ) T cells expressed as a percentage of CD4+ cells and absolute number. (F) GC T FH cells (CD4+, CXCR5+, PD-1 hi ) presented as frequency among the CD4+ population and absolute number. (G) PD-1 expression level in CD4+ cells assessed by FACS. (H) GC B cell (B220+, GL7+, IgD−) presented as frequency among the B220+ population and absolute number. (I) Percentage and absolute number of plasma cells (B220 low , CD138 hi ). Each symbol represents an individual mouse. Bars indicate the mean±SEM N.S., not significant; *p

    Journal: Annals of the Rheumatic Diseases

    Article Title: B cell OX40L supports T follicular helper cell development and contributes to SLE pathogenesis

    doi: 10.1136/annrheumdis-2017-211499

    Figure Lengend Snippet: OX40L deficiency ameliorates the phenotype of B6. Sle16 lupus-prone mice. Comparison between female B6. Sle16 and B6.Sle16.Tnfsf4 −/− female mice at 9 months of age. (A) Quantitation of spleen/body weight ratio and spleen weight. (B) Absolute number of cells per spleen. (C) Serum level of IgG and IgM at 6 and 9 months. (D) Titre of IgM anti-dsDNA and anti-ssDNA. (E) Quantitation of naïve (T N ) (CD4+, CD62L+, CD44 low ), (T EFF ) effector (CD4+, CD62L low , CD44 low ), T EM effector/memory (CD4+, CD62L low/neg , CD44 hi ) and T CM central/memory (CD4+, CD62L+, CD44 hi ) T cells expressed as a percentage of CD4+ cells and absolute number. (F) GC T FH cells (CD4+, CXCR5+, PD-1 hi ) presented as frequency among the CD4+ population and absolute number. (G) PD-1 expression level in CD4+ cells assessed by FACS. (H) GC B cell (B220+, GL7+, IgD−) presented as frequency among the B220+ population and absolute number. (I) Percentage and absolute number of plasma cells (B220 low , CD138 hi ). Each symbol represents an individual mouse. Bars indicate the mean±SEM N.S., not significant; *p

    Article Snippet: Bound Abs were detected with AP-conjugated goat anti-mouse IgG (-chain specific) (Sigma-Aldrich) or IgM (Southern Biotechnology Associates).

    Techniques: Mouse Assay, Quantitation Assay, Expressing, FACS

    rPyM2-MAEBL-specific antibodies generated in vaccinated mice recognize the native protein expressed by P. yoelii . Fixed IFA slides of mature P. yoelii schizonts were incubated with pooled sera from mice immunized as indicated in a 1:100 dilution. Bound IgG was stained with Alexa Fluor 568, and the parasite nuclei were stained with DAPI.

    Journal: Infection and Immunity

    Article Title: Immunization with the MAEBL M2 Domain Protects against Lethal Plasmodium yoelii Infection

    doi: 10.1128/IAI.00262-15

    Figure Lengend Snippet: rPyM2-MAEBL-specific antibodies generated in vaccinated mice recognize the native protein expressed by P. yoelii . Fixed IFA slides of mature P. yoelii schizonts were incubated with pooled sera from mice immunized as indicated in a 1:100 dilution. Bound IgG was stained with Alexa Fluor 568, and the parasite nuclei were stained with DAPI.

    Article Snippet: A 1:2,000 dilution of peroxidase-coupled goat anti-mouse IgG (Sigma) conjugated with Alexa Fluor 568 was used as the secondary antibody.

    Techniques: Generated, Mouse Assay, Immunofluorescence, Incubation, Staining

    Susceptibility of target cells for FasL mediated cell death. ( A ) CEA + LS174T, SW948 and H508 tumor cells and CEA − Jurkat cells for control were incubated in 96-well micro-titer plates in the presence of recombinant CD8 -Fas-L fusion protein (2 µg/mL) for 24 h. The viability of tumor cells was determined by the XTT assay and specific cytolysis calculated. Data represent mean of technical replicates + SD. Significant differences were calculated by the Student’s t test. A representative experiment out of three is shown. ( B ) Resting CD4 + CD25 − T cells express low levels of perforin. CD4 + CD25 − CAR T cells were cultured without stimulation for 48 h and tested by flow cytometry for intracellular perforin levels. An isotype matched mAb served as control for staining. ( C , D ) Perforin and granzyme B were upregulated in CD4 + CD25 − but not in CD4 + CD25 + CAR T cells upon CAR stimulation. CD4 + CD25 − and CD4 + CD25 + CAR T cells (2.5 × 10 4 /well) were stimulated in micro-titer plates coated with 10 µg/mL anti-idiotypic mAb BW2064 or with an isotype control IgG1 for 48 h. Cells from triplicates were pooled, stained with anti-perforin ( C ) or anti-granzyme B ( D ) mAbs, and recorded by intracellular flow cytometry.

    Journal: Cancers

    Article Title: Most Do, but Some Do Not: CD4+CD25− T Cells, but Not CD4+CD25+ Treg Cells, Are Cytolytic When Redirected by a Chimeric Antigen Receptor (CAR)

    doi: 10.3390/cancers9090112

    Figure Lengend Snippet: Susceptibility of target cells for FasL mediated cell death. ( A ) CEA + LS174T, SW948 and H508 tumor cells and CEA − Jurkat cells for control were incubated in 96-well micro-titer plates in the presence of recombinant CD8 -Fas-L fusion protein (2 µg/mL) for 24 h. The viability of tumor cells was determined by the XTT assay and specific cytolysis calculated. Data represent mean of technical replicates + SD. Significant differences were calculated by the Student’s t test. A representative experiment out of three is shown. ( B ) Resting CD4 + CD25 − T cells express low levels of perforin. CD4 + CD25 − CAR T cells were cultured without stimulation for 48 h and tested by flow cytometry for intracellular perforin levels. An isotype matched mAb served as control for staining. ( C , D ) Perforin and granzyme B were upregulated in CD4 + CD25 − but not in CD4 + CD25 + CAR T cells upon CAR stimulation. CD4 + CD25 − and CD4 + CD25 + CAR T cells (2.5 × 10 4 /well) were stimulated in micro-titer plates coated with 10 µg/mL anti-idiotypic mAb BW2064 or with an isotype control IgG1 for 48 h. Cells from triplicates were pooled, stained with anti-perforin ( C ) or anti-granzyme B ( D ) mAbs, and recorded by intracellular flow cytometry.

    Article Snippet: MAbs were affinity purified from supernatants utilizing an agarose immobilized goat anti-mouse IgG1 (Sigma, Deisenhofen, Germany) or a sepharose (Amersham Pharmacia, Freiburg, Germany) immobilized goat anti-mouse IgG2a antibody (Southern Biotechnology, Birmingham, AL, USA).

    Techniques: Incubation, Recombinant, XTT Assay, Cell Culture, Flow Cytometry, Cytometry, Staining

    CD4 + T cells release a distinctive set of cytokines upon CAR mediated activation. ( A ) CD4 + CD25 − and CD4 + CD25 + anti-CEA CAR T cells (10 4 /well) were incubated in micro-titer plates coated with serial dilutions of the CAR specific anti-idiotypic monoclonal antibody (mAb) BW2064 or an IgG1 isotype control mAb (0.01–10 µg/mL) and cultivated for 48 h. Supernatants were recorded for cytokines by ELISA. Data represent the mean of technical replicates ± SD. Significant differences were calculated by the Student’s t test and significant data ( p

    Journal: Cancers

    Article Title: Most Do, but Some Do Not: CD4+CD25− T Cells, but Not CD4+CD25+ Treg Cells, Are Cytolytic When Redirected by a Chimeric Antigen Receptor (CAR)

    doi: 10.3390/cancers9090112

    Figure Lengend Snippet: CD4 + T cells release a distinctive set of cytokines upon CAR mediated activation. ( A ) CD4 + CD25 − and CD4 + CD25 + anti-CEA CAR T cells (10 4 /well) were incubated in micro-titer plates coated with serial dilutions of the CAR specific anti-idiotypic monoclonal antibody (mAb) BW2064 or an IgG1 isotype control mAb (0.01–10 µg/mL) and cultivated for 48 h. Supernatants were recorded for cytokines by ELISA. Data represent the mean of technical replicates ± SD. Significant differences were calculated by the Student’s t test and significant data ( p

    Article Snippet: MAbs were affinity purified from supernatants utilizing an agarose immobilized goat anti-mouse IgG1 (Sigma, Deisenhofen, Germany) or a sepharose (Amersham Pharmacia, Freiburg, Germany) immobilized goat anti-mouse IgG2a antibody (Southern Biotechnology, Birmingham, AL, USA).

    Techniques: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay

    Isolation of class II−restricted CD4 + cytotoxic T cells (CD4 + T) cell subsets. ( A ) Peripheral blood lymphocytes were isolated from healthy donors and fractionated by magnetic cell sorting procedures into the CD4 + CD25 − and CD4 + CD25 + populations. Isolated cells were stained with anti-CD4 and anti-CD25 antibodies and analyzed by flow cytometry. ( B , C ) Expression of chimeric antigen receptors (CARs) in CD4 + CD25 + and CD4 + CD25 − T cells. Isolated CD4 + CD25 − and CD4 + CD25 + T cells were activated, expanded and engineered with an anti-carcinoembryonic antigen (CEA) CD28−ζ CAR as described in Materials and Methods. Modified cells were identified by staining with an anti-CD3 and anti-IgG antibody, which recognizes the extracellular IgG1 spacer within the CAR, and analyzed by flow cytometry. ( B ) Dot blots of a flow cytometric analysis of blood cells from a representative healthy donor. ( C ) Summary of mean fluorescence intensity (mfi) and number of CAR + T cells in CD4 + CD25 + and CD4 + CD25 − T cells from healthy donors ( n = 5). Numbers represent mean values ± Standard Deviation (SD). Significance was calculated by the Student’s t test.

    Journal: Cancers

    Article Title: Most Do, but Some Do Not: CD4+CD25− T Cells, but Not CD4+CD25+ Treg Cells, Are Cytolytic When Redirected by a Chimeric Antigen Receptor (CAR)

    doi: 10.3390/cancers9090112

    Figure Lengend Snippet: Isolation of class II−restricted CD4 + cytotoxic T cells (CD4 + T) cell subsets. ( A ) Peripheral blood lymphocytes were isolated from healthy donors and fractionated by magnetic cell sorting procedures into the CD4 + CD25 − and CD4 + CD25 + populations. Isolated cells were stained with anti-CD4 and anti-CD25 antibodies and analyzed by flow cytometry. ( B , C ) Expression of chimeric antigen receptors (CARs) in CD4 + CD25 + and CD4 + CD25 − T cells. Isolated CD4 + CD25 − and CD4 + CD25 + T cells were activated, expanded and engineered with an anti-carcinoembryonic antigen (CEA) CD28−ζ CAR as described in Materials and Methods. Modified cells were identified by staining with an anti-CD3 and anti-IgG antibody, which recognizes the extracellular IgG1 spacer within the CAR, and analyzed by flow cytometry. ( B ) Dot blots of a flow cytometric analysis of blood cells from a representative healthy donor. ( C ) Summary of mean fluorescence intensity (mfi) and number of CAR + T cells in CD4 + CD25 + and CD4 + CD25 − T cells from healthy donors ( n = 5). Numbers represent mean values ± Standard Deviation (SD). Significance was calculated by the Student’s t test.

    Article Snippet: MAbs were affinity purified from supernatants utilizing an agarose immobilized goat anti-mouse IgG1 (Sigma, Deisenhofen, Germany) or a sepharose (Amersham Pharmacia, Freiburg, Germany) immobilized goat anti-mouse IgG2a antibody (Southern Biotechnology, Birmingham, AL, USA).

    Techniques: Isolation, FACS, Staining, Flow Cytometry, Cytometry, Expressing, Modification, Fluorescence, Standard Deviation

    Serum anti-rTsCB IgG titers determined by rTsCB-ELISA. The OD values are shown as the mean ± SD of anti-rTsCB IgG levels of 20 immunized mice

    Journal: Parasites & Vectors

    Article Title: Vaccination of mice with a recombinant novel cathepsin B inhibits Trichinella spiralis development, reduces the fecundity and worm burden

    doi: 10.1186/s13071-019-3833-9

    Figure Lengend Snippet: Serum anti-rTsCB IgG titers determined by rTsCB-ELISA. The OD values are shown as the mean ± SD of anti-rTsCB IgG levels of 20 immunized mice

    Article Snippet: After washing, the plate was blocked using 5% skimmed milk in PBST, then probed with mouse immune sera (1:100) at 37 °C for 1 h. Goat anti-mouse IgG (IgG1 and IgG2a)-HRP conjugates (1:5000; Sigma-Aldrich, St. Louis, MO, USA) or Goat anti-mouse IgE-HRP conjugate (1:2500; Southern Biotech, Tuscaloosa, AL, USA) were added and incubated for 1 h at 37 °C.

    Techniques: Enzyme-linked Immunosorbent Assay, Mouse Assay

    Specific antibody response in mice immunized with rTsCB. a Specific total IgG in mice immunized with rTsCB or control mice (adjuvant and PBS) at different time intervals following vaccination. Specific IgG1 ( b ) and IgG2a ( c ) subclass responses against rTsCB at different time points following vaccination. d Specific IgE level in vaccinated mice. The OD values are shown as the mean ± SD of antibody levels ( n = 10). Vaccination time point is indicated with an arrow. * P

    Journal: Parasites & Vectors

    Article Title: Vaccination of mice with a recombinant novel cathepsin B inhibits Trichinella spiralis development, reduces the fecundity and worm burden

    doi: 10.1186/s13071-019-3833-9

    Figure Lengend Snippet: Specific antibody response in mice immunized with rTsCB. a Specific total IgG in mice immunized with rTsCB or control mice (adjuvant and PBS) at different time intervals following vaccination. Specific IgG1 ( b ) and IgG2a ( c ) subclass responses against rTsCB at different time points following vaccination. d Specific IgE level in vaccinated mice. The OD values are shown as the mean ± SD of antibody levels ( n = 10). Vaccination time point is indicated with an arrow. * P

    Article Snippet: After washing, the plate was blocked using 5% skimmed milk in PBST, then probed with mouse immune sera (1:100) at 37 °C for 1 h. Goat anti-mouse IgG (IgG1 and IgG2a)-HRP conjugates (1:5000; Sigma-Aldrich, St. Louis, MO, USA) or Goat anti-mouse IgE-HRP conjugate (1:2500; Southern Biotech, Tuscaloosa, AL, USA) were added and incubated for 1 h at 37 °C.

    Techniques: Mouse Assay

    Surface expression of β3 and αV integrin subunits in M. tuberculosis –stimulated monocytes adherent to ECM proteins. Monocytes cultured in the presence or absence of type I collagen or fibronectin were stimulated for 24 h with CoMtb (1:5 dilution), and CoMCont as control, or directly infected with M. tuberculosis (MOI = 1). Cells were fixed, blocked, and stained with FITC-conjugated anti-integrin β1or primary anti-integrin β2, β3, or αV Abs and secondary FITC-conjugated anti-mouse IgG Ab. Secondary Ab alone or FITC-conjugated IgG1 isotype Ab were used as controls. ( A ) Histograms of integrin subunits β1, β2, and β3 for control and CoMtb-stimulated monocytes show an increase in β3 expression with CoMtb stimulation. MFIs of integrin subunits ( B ) β3 and ( C ) αV in monocytes stimulated with M. tuberculosis , and MFIs of ( D ) β3 and ( E ) αV in monocytes stimulated with CoMtb, in the presence or absence of matrix components ( n = 4). MFIs were normalized to baseline MFIs of respective controls (control media or CoMCont). (B)–(E) show data points and means. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Monocyte Adhesion, Migration, and Extracellular Matrix Breakdown Are Regulated by Integrin αVβ3 in Mycobacterium tuberculosis Infection

    doi: 10.4049/jimmunol.1700128

    Figure Lengend Snippet: Surface expression of β3 and αV integrin subunits in M. tuberculosis –stimulated monocytes adherent to ECM proteins. Monocytes cultured in the presence or absence of type I collagen or fibronectin were stimulated for 24 h with CoMtb (1:5 dilution), and CoMCont as control, or directly infected with M. tuberculosis (MOI = 1). Cells were fixed, blocked, and stained with FITC-conjugated anti-integrin β1or primary anti-integrin β2, β3, or αV Abs and secondary FITC-conjugated anti-mouse IgG Ab. Secondary Ab alone or FITC-conjugated IgG1 isotype Ab were used as controls. ( A ) Histograms of integrin subunits β1, β2, and β3 for control and CoMtb-stimulated monocytes show an increase in β3 expression with CoMtb stimulation. MFIs of integrin subunits ( B ) β3 and ( C ) αV in monocytes stimulated with M. tuberculosis , and MFIs of ( D ) β3 and ( E ) αV in monocytes stimulated with CoMtb, in the presence or absence of matrix components ( n = 4). MFIs were normalized to baseline MFIs of respective controls (control media or CoMCont). (B)–(E) show data points and means. * p

    Article Snippet: FITC-conjugated goat anti-mouse IgG1 (Sigma-Aldrich, Dorset, U.K.), and Cy5 conjugated goat anti-rabbit (Abcam, Cambridge, U.K.) were used as secondary Abs.

    Techniques: Expressing, Cell Culture, Infection, Staining

    Integrin αVβ3 upregulates type I collagen breakdown and increased monocyte adhesion and transmigration in M. tuberculosis infection. Slides were precoated with DQ type I collagen in which FITC is quenched and fluorescence is released only in areas of collagen degradation. Monocytes were preincubated with or without a function blocking anti-integrin β3 Ab or anti-IgG1 isotype control, followed by M. tuberculosis infection (MOI = 1). ( A ) Panels show brightfield, DAPI nuclear counterstain (blue), collagen degradation (green), and merged images. Scale bar, 25 μm. ( B ) CoMtb stimulation increased monocyte adhesion compared with CoMCont, which was blocked by inhibition of integrin β3. Next 1 × 10 5 monocytes per well were prestained with CellTracker Green CMFDA dye were stimulated with CoMtb or CoMCont. Integrin β3–mediated adhesion was inhibited with anti-integrin β3 Ab. Control monocytes preincubated with IgG1 isotype Abs. ( C ) Monocyte migration is increased with CoMtb stimulation, which is blocked with inhibition of integrin αV or β3. Transwells were precoated with type I collagen, and CoMtb or CoMCont was added to the basal side in a 1:2 dilution. Integrins were blocked with anti-integrin αV and β3 Abs, or an IgG1 isotype control Ab. Bars show mean ± SD. Data are representative of three independent experiments performed in triplicate. *** p

    Journal: The Journal of Immunology Author Choice

    Article Title: Monocyte Adhesion, Migration, and Extracellular Matrix Breakdown Are Regulated by Integrin αVβ3 in Mycobacterium tuberculosis Infection

    doi: 10.4049/jimmunol.1700128

    Figure Lengend Snippet: Integrin αVβ3 upregulates type I collagen breakdown and increased monocyte adhesion and transmigration in M. tuberculosis infection. Slides were precoated with DQ type I collagen in which FITC is quenched and fluorescence is released only in areas of collagen degradation. Monocytes were preincubated with or without a function blocking anti-integrin β3 Ab or anti-IgG1 isotype control, followed by M. tuberculosis infection (MOI = 1). ( A ) Panels show brightfield, DAPI nuclear counterstain (blue), collagen degradation (green), and merged images. Scale bar, 25 μm. ( B ) CoMtb stimulation increased monocyte adhesion compared with CoMCont, which was blocked by inhibition of integrin β3. Next 1 × 10 5 monocytes per well were prestained with CellTracker Green CMFDA dye were stimulated with CoMtb or CoMCont. Integrin β3–mediated adhesion was inhibited with anti-integrin β3 Ab. Control monocytes preincubated with IgG1 isotype Abs. ( C ) Monocyte migration is increased with CoMtb stimulation, which is blocked with inhibition of integrin αV or β3. Transwells were precoated with type I collagen, and CoMtb or CoMCont was added to the basal side in a 1:2 dilution. Integrins were blocked with anti-integrin αV and β3 Abs, or an IgG1 isotype control Ab. Bars show mean ± SD. Data are representative of three independent experiments performed in triplicate. *** p

    Article Snippet: FITC-conjugated goat anti-mouse IgG1 (Sigma-Aldrich, Dorset, U.K.), and Cy5 conjugated goat anti-rabbit (Abcam, Cambridge, U.K.) were used as secondary Abs.

    Techniques: Transmigration Assay, Infection, Fluorescence, Blocking Assay, Inhibition, Migration

    Specific antibody response in mice immunized with untargeted (SAG1t) or targeted (SA2) proteins. Detection of specific anti-SAG1 IgG antibodies (A) and IgG subclasses (B) in sera of mice (12/group) primed and boosted twice with SAG1t, SA2, or phosphate-buffered saline formulated with Poly I:C by combined routes. Serum samples collected after the last boost were tested by ELISA using SAG1t protein as the coating antigen. Results are expressed as the mean ± SEM ( n = 12) of log2 titers and represent one of two independent experiments. *** P

    Journal: Frontiers in Immunology

    Article Title: Targeted Delivery of Toxoplasma gondii Antigens to Dendritic Cells Promote Immunogenicity and Protective Efficiency against Toxoplasmosis

    doi: 10.3389/fimmu.2018.00317

    Figure Lengend Snippet: Specific antibody response in mice immunized with untargeted (SAG1t) or targeted (SA2) proteins. Detection of specific anti-SAG1 IgG antibodies (A) and IgG subclasses (B) in sera of mice (12/group) primed and boosted twice with SAG1t, SA2, or phosphate-buffered saline formulated with Poly I:C by combined routes. Serum samples collected after the last boost were tested by ELISA using SAG1t protein as the coating antigen. Results are expressed as the mean ± SEM ( n = 12) of log2 titers and represent one of two independent experiments. *** P

    Article Snippet: The plates were then washed again and incubated for 1 h at 37°C with Goat anti-Mouse IgG alkaline phosphatase (1:5,000, Sigma), rat anti-mouse IgG1 alkaline phosphatase (X56), or rat anti-mouse IgG2a alkaline phosphatase (R19-15) (both at 1:1,000, BD Pharmingen).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay