alexa fluor 647-conjugated goat anti-mouse igg antibody Thermo Fisher Search Results


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  • 99
    Thermo Fisher goat anti mouse igg h l cross adsorbed secondary antibody
    Goat Anti Mouse Igg H L Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14005 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 647 conjugated goat anti mouse igg
    Infectivity of recombinant MP-12 encoding a glutamine (Q) in place of an asparagine (N) at N-X-S/T sequon(s) in Gn or Gc. ( A ) Jurkat-DC-SIGN cells stably co-expressing green fluorescent protein (GFP) and human DC-SIGN (~17% in cell population) (top panels), or parental Jurkat cells, which express neither DC-SIGN nor GFP (bottom panels), were mock-infected (left and center panels) or infected with rMP-12 (right panels) at a multiplicity of infection (MOI) of 3.6. At 6 hpi, cells were fixed, permeabilized, and then stained with a cocktail of mouse anti-RVFV antibody and <t>Alexa</t> Fluor 488-conjugated rabbit anti-GFP antibody, or a cocktail of mouse anti-RVFV antibody and Alexa Fluor 488-conjugated normal rabbit <t>IgG.</t> Subsequently, cells were stained with Alexa Fluor 647-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. Since permeabilized Jurkat-DC-SIGN cells showed poor intrinsic GFP signals, as shown with Alexa Fluor 488-conjugated normal rabbit IgG (left top panel), rabbit anti-GFP antibody (center and right top panels) was used to detect GFP signals of permeabilized Jurkat-DC-SIGN cells. Q1, GFP-negative (DC-SIGN-negative) and RVFV-infected cell population; Q2, GFP-positive (DC-SIGN-positive) and RVFV-infected cell population; Q3, GFP-positive (DC-SIGN-positive) and uninfected cell population; Q4, GFP-negative (DC-SIGN-negative) and uninfected cell population; ( B , C ) Jurkat-DC-SIGN cells were infected with rMP-12 or that lacking one ( B ) or two sequons ( C ). Relative number of RVFV-infected cells in the GFP-positive cell population (Q2/(Q2+Q3)) normalized to that of RVFV-infected cells in the GFP-negative cell population (Q1/(Q1+Q4)) are shown. Graphs represent mean + standard deviations for three independent experiments.
    Alexa Fluor 647 Conjugated Goat Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 409 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse igg h l highly cross adsorbed secondary antibody
    Infectivity of recombinant MP-12 encoding a glutamine (Q) in place of an asparagine (N) at N-X-S/T sequon(s) in Gn or Gc. ( A ) Jurkat-DC-SIGN cells stably co-expressing green fluorescent protein (GFP) and human DC-SIGN (~17% in cell population) (top panels), or parental Jurkat cells, which express neither DC-SIGN nor GFP (bottom panels), were mock-infected (left and center panels) or infected with rMP-12 (right panels) at a multiplicity of infection (MOI) of 3.6. At 6 hpi, cells were fixed, permeabilized, and then stained with a cocktail of mouse anti-RVFV antibody and <t>Alexa</t> Fluor 488-conjugated rabbit anti-GFP antibody, or a cocktail of mouse anti-RVFV antibody and Alexa Fluor 488-conjugated normal rabbit <t>IgG.</t> Subsequently, cells were stained with Alexa Fluor 647-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. Since permeabilized Jurkat-DC-SIGN cells showed poor intrinsic GFP signals, as shown with Alexa Fluor 488-conjugated normal rabbit IgG (left top panel), rabbit anti-GFP antibody (center and right top panels) was used to detect GFP signals of permeabilized Jurkat-DC-SIGN cells. Q1, GFP-negative (DC-SIGN-negative) and RVFV-infected cell population; Q2, GFP-positive (DC-SIGN-positive) and RVFV-infected cell population; Q3, GFP-positive (DC-SIGN-positive) and uninfected cell population; Q4, GFP-negative (DC-SIGN-negative) and uninfected cell population; ( B , C ) Jurkat-DC-SIGN cells were infected with rMP-12 or that lacking one ( B ) or two sequons ( C ). Relative number of RVFV-infected cells in the GFP-positive cell population (Q2/(Q2+Q3)) normalized to that of RVFV-infected cells in the GFP-negative cell population (Q1/(Q1+Q4)) are shown. Graphs represent mean + standard deviations for three independent experiments.
    Goat Anti Mouse Igg H L Highly Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9004 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa647 conjugated goat anti mouse igg2a antibodies
    Infectivity of recombinant MP-12 encoding a glutamine (Q) in place of an asparagine (N) at N-X-S/T sequon(s) in Gn or Gc. ( A ) Jurkat-DC-SIGN cells stably co-expressing green fluorescent protein (GFP) and human DC-SIGN (~17% in cell population) (top panels), or parental Jurkat cells, which express neither DC-SIGN nor GFP (bottom panels), were mock-infected (left and center panels) or infected with rMP-12 (right panels) at a multiplicity of infection (MOI) of 3.6. At 6 hpi, cells were fixed, permeabilized, and then stained with a cocktail of mouse anti-RVFV antibody and <t>Alexa</t> Fluor 488-conjugated rabbit anti-GFP antibody, or a cocktail of mouse anti-RVFV antibody and Alexa Fluor 488-conjugated normal rabbit <t>IgG.</t> Subsequently, cells were stained with Alexa Fluor 647-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. Since permeabilized Jurkat-DC-SIGN cells showed poor intrinsic GFP signals, as shown with Alexa Fluor 488-conjugated normal rabbit IgG (left top panel), rabbit anti-GFP antibody (center and right top panels) was used to detect GFP signals of permeabilized Jurkat-DC-SIGN cells. Q1, GFP-negative (DC-SIGN-negative) and RVFV-infected cell population; Q2, GFP-positive (DC-SIGN-positive) and RVFV-infected cell population; Q3, GFP-positive (DC-SIGN-positive) and uninfected cell population; Q4, GFP-negative (DC-SIGN-negative) and uninfected cell population; ( B , C ) Jurkat-DC-SIGN cells were infected with rMP-12 or that lacking one ( B ) or two sequons ( C ). Relative number of RVFV-infected cells in the GFP-positive cell population (Q2/(Q2+Q3)) normalized to that of RVFV-infected cells in the GFP-negative cell population (Q1/(Q1+Q4)) are shown. Graphs represent mean + standard deviations for three independent experiments.
    Alexa647 Conjugated Goat Anti Mouse Igg2a Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse igg1 cross adsorbed secondary antibody
    Infectivity of recombinant MP-12 encoding a glutamine (Q) in place of an asparagine (N) at N-X-S/T sequon(s) in Gn or Gc. ( A ) Jurkat-DC-SIGN cells stably co-expressing green fluorescent protein (GFP) and human DC-SIGN (~17% in cell population) (top panels), or parental Jurkat cells, which express neither DC-SIGN nor GFP (bottom panels), were mock-infected (left and center panels) or infected with rMP-12 (right panels) at a multiplicity of infection (MOI) of 3.6. At 6 hpi, cells were fixed, permeabilized, and then stained with a cocktail of mouse anti-RVFV antibody and <t>Alexa</t> Fluor 488-conjugated rabbit anti-GFP antibody, or a cocktail of mouse anti-RVFV antibody and Alexa Fluor 488-conjugated normal rabbit <t>IgG.</t> Subsequently, cells were stained with Alexa Fluor 647-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. Since permeabilized Jurkat-DC-SIGN cells showed poor intrinsic GFP signals, as shown with Alexa Fluor 488-conjugated normal rabbit IgG (left top panel), rabbit anti-GFP antibody (center and right top panels) was used to detect GFP signals of permeabilized Jurkat-DC-SIGN cells. Q1, GFP-negative (DC-SIGN-negative) and RVFV-infected cell population; Q2, GFP-positive (DC-SIGN-positive) and RVFV-infected cell population; Q3, GFP-positive (DC-SIGN-positive) and uninfected cell population; Q4, GFP-negative (DC-SIGN-negative) and uninfected cell population; ( B , C ) Jurkat-DC-SIGN cells were infected with rMP-12 or that lacking one ( B ) or two sequons ( C ). Relative number of RVFV-infected cells in the GFP-positive cell population (Q2/(Q2+Q3)) normalized to that of RVFV-infected cells in the GFP-negative cell population (Q1/(Q1+Q4)) are shown. Graphs represent mean + standard deviations for three independent experiments.
    Goat Anti Mouse Igg1 Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse igg alexa fluor 647 conjugate
    Infectivity of recombinant MP-12 encoding a glutamine (Q) in place of an asparagine (N) at N-X-S/T sequon(s) in Gn or Gc. ( A ) Jurkat-DC-SIGN cells stably co-expressing green fluorescent protein (GFP) and human DC-SIGN (~17% in cell population) (top panels), or parental Jurkat cells, which express neither DC-SIGN nor GFP (bottom panels), were mock-infected (left and center panels) or infected with rMP-12 (right panels) at a multiplicity of infection (MOI) of 3.6. At 6 hpi, cells were fixed, permeabilized, and then stained with a cocktail of mouse anti-RVFV antibody and <t>Alexa</t> Fluor 488-conjugated rabbit anti-GFP antibody, or a cocktail of mouse anti-RVFV antibody and Alexa Fluor 488-conjugated normal rabbit <t>IgG.</t> Subsequently, cells were stained with Alexa Fluor 647-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. Since permeabilized Jurkat-DC-SIGN cells showed poor intrinsic GFP signals, as shown with Alexa Fluor 488-conjugated normal rabbit IgG (left top panel), rabbit anti-GFP antibody (center and right top panels) was used to detect GFP signals of permeabilized Jurkat-DC-SIGN cells. Q1, GFP-negative (DC-SIGN-negative) and RVFV-infected cell population; Q2, GFP-positive (DC-SIGN-positive) and RVFV-infected cell population; Q3, GFP-positive (DC-SIGN-positive) and uninfected cell population; Q4, GFP-negative (DC-SIGN-negative) and uninfected cell population; ( B , C ) Jurkat-DC-SIGN cells were infected with rMP-12 or that lacking one ( B ) or two sequons ( C ). Relative number of RVFV-infected cells in the GFP-positive cell population (Q2/(Q2+Q3)) normalized to that of RVFV-infected cells in the GFP-negative cell population (Q1/(Q1+Q4)) are shown. Graphs represent mean + standard deviations for three independent experiments.
    Goat Anti Mouse Igg Alexa Fluor 647 Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse igg h l superclonal secondary antibody
    Infectivity of recombinant MP-12 encoding a glutamine (Q) in place of an asparagine (N) at N-X-S/T sequon(s) in Gn or Gc. ( A ) Jurkat-DC-SIGN cells stably co-expressing green fluorescent protein (GFP) and human DC-SIGN (~17% in cell population) (top panels), or parental Jurkat cells, which express neither DC-SIGN nor GFP (bottom panels), were mock-infected (left and center panels) or infected with rMP-12 (right panels) at a multiplicity of infection (MOI) of 3.6. At 6 hpi, cells were fixed, permeabilized, and then stained with a cocktail of mouse anti-RVFV antibody and <t>Alexa</t> Fluor 488-conjugated rabbit anti-GFP antibody, or a cocktail of mouse anti-RVFV antibody and Alexa Fluor 488-conjugated normal rabbit <t>IgG.</t> Subsequently, cells were stained with Alexa Fluor 647-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. Since permeabilized Jurkat-DC-SIGN cells showed poor intrinsic GFP signals, as shown with Alexa Fluor 488-conjugated normal rabbit IgG (left top panel), rabbit anti-GFP antibody (center and right top panels) was used to detect GFP signals of permeabilized Jurkat-DC-SIGN cells. Q1, GFP-negative (DC-SIGN-negative) and RVFV-infected cell population; Q2, GFP-positive (DC-SIGN-positive) and RVFV-infected cell population; Q3, GFP-positive (DC-SIGN-positive) and uninfected cell population; Q4, GFP-negative (DC-SIGN-negative) and uninfected cell population; ( B , C ) Jurkat-DC-SIGN cells were infected with rMP-12 or that lacking one ( B ) or two sequons ( C ). Relative number of RVFV-infected cells in the GFP-positive cell population (Q2/(Q2+Q3)) normalized to that of RVFV-infected cells in the GFP-negative cell population (Q1/(Q1+Q4)) are shown. Graphs represent mean + standard deviations for three independent experiments.
    Goat Anti Mouse Igg H L Superclonal Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 411 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher secondary antibodies
    Infectivity of recombinant MP-12 encoding a glutamine (Q) in place of an asparagine (N) at N-X-S/T sequon(s) in Gn or Gc. ( A ) Jurkat-DC-SIGN cells stably co-expressing green fluorescent protein (GFP) and human DC-SIGN (~17% in cell population) (top panels), or parental Jurkat cells, which express neither DC-SIGN nor GFP (bottom panels), were mock-infected (left and center panels) or infected with rMP-12 (right panels) at a multiplicity of infection (MOI) of 3.6. At 6 hpi, cells were fixed, permeabilized, and then stained with a cocktail of mouse anti-RVFV antibody and <t>Alexa</t> Fluor 488-conjugated rabbit anti-GFP antibody, or a cocktail of mouse anti-RVFV antibody and Alexa Fluor 488-conjugated normal rabbit <t>IgG.</t> Subsequently, cells were stained with Alexa Fluor 647-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. Since permeabilized Jurkat-DC-SIGN cells showed poor intrinsic GFP signals, as shown with Alexa Fluor 488-conjugated normal rabbit IgG (left top panel), rabbit anti-GFP antibody (center and right top panels) was used to detect GFP signals of permeabilized Jurkat-DC-SIGN cells. Q1, GFP-negative (DC-SIGN-negative) and RVFV-infected cell population; Q2, GFP-positive (DC-SIGN-positive) and RVFV-infected cell population; Q3, GFP-positive (DC-SIGN-positive) and uninfected cell population; Q4, GFP-negative (DC-SIGN-negative) and uninfected cell population; ( B , C ) Jurkat-DC-SIGN cells were infected with rMP-12 or that lacking one ( B ) or two sequons ( C ). Relative number of RVFV-infected cells in the GFP-positive cell population (Q2/(Q2+Q3)) normalized to that of RVFV-infected cells in the GFP-negative cell population (Q1/(Q1+Q4)) are shown. Graphs represent mean + standard deviations for three independent experiments.
    Secondary Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16006 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 647 conjugated f
    Infectivity of recombinant MP-12 encoding a glutamine (Q) in place of an asparagine (N) at N-X-S/T sequon(s) in Gn or Gc. ( A ) Jurkat-DC-SIGN cells stably co-expressing green fluorescent protein (GFP) and human DC-SIGN (~17% in cell population) (top panels), or parental Jurkat cells, which express neither DC-SIGN nor GFP (bottom panels), were mock-infected (left and center panels) or infected with rMP-12 (right panels) at a multiplicity of infection (MOI) of 3.6. At 6 hpi, cells were fixed, permeabilized, and then stained with a cocktail of mouse anti-RVFV antibody and <t>Alexa</t> Fluor 488-conjugated rabbit anti-GFP antibody, or a cocktail of mouse anti-RVFV antibody and Alexa Fluor 488-conjugated normal rabbit <t>IgG.</t> Subsequently, cells were stained with Alexa Fluor 647-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. Since permeabilized Jurkat-DC-SIGN cells showed poor intrinsic GFP signals, as shown with Alexa Fluor 488-conjugated normal rabbit IgG (left top panel), rabbit anti-GFP antibody (center and right top panels) was used to detect GFP signals of permeabilized Jurkat-DC-SIGN cells. Q1, GFP-negative (DC-SIGN-negative) and RVFV-infected cell population; Q2, GFP-positive (DC-SIGN-positive) and RVFV-infected cell population; Q3, GFP-positive (DC-SIGN-positive) and uninfected cell population; Q4, GFP-negative (DC-SIGN-negative) and uninfected cell population; ( B , C ) Jurkat-DC-SIGN cells were infected with rMP-12 or that lacking one ( B ) or two sequons ( C ). Relative number of RVFV-infected cells in the GFP-positive cell population (Q2/(Q2+Q3)) normalized to that of RVFV-infected cells in the GFP-negative cell population (Q1/(Q1+Q4)) are shown. Graphs represent mean + standard deviations for three independent experiments.
    Alexa Fluor 647 Conjugated F, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 952 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti mouse igg conjugated to alexa fluor 647
    Infectivity of recombinant MP-12 encoding a glutamine (Q) in place of an asparagine (N) at N-X-S/T sequon(s) in Gn or Gc. ( A ) Jurkat-DC-SIGN cells stably co-expressing green fluorescent protein (GFP) and human DC-SIGN (~17% in cell population) (top panels), or parental Jurkat cells, which express neither DC-SIGN nor GFP (bottom panels), were mock-infected (left and center panels) or infected with rMP-12 (right panels) at a multiplicity of infection (MOI) of 3.6. At 6 hpi, cells were fixed, permeabilized, and then stained with a cocktail of mouse anti-RVFV antibody and <t>Alexa</t> Fluor 488-conjugated rabbit anti-GFP antibody, or a cocktail of mouse anti-RVFV antibody and Alexa Fluor 488-conjugated normal rabbit <t>IgG.</t> Subsequently, cells were stained with Alexa Fluor 647-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. Since permeabilized Jurkat-DC-SIGN cells showed poor intrinsic GFP signals, as shown with Alexa Fluor 488-conjugated normal rabbit IgG (left top panel), rabbit anti-GFP antibody (center and right top panels) was used to detect GFP signals of permeabilized Jurkat-DC-SIGN cells. Q1, GFP-negative (DC-SIGN-negative) and RVFV-infected cell population; Q2, GFP-positive (DC-SIGN-positive) and RVFV-infected cell population; Q3, GFP-positive (DC-SIGN-positive) and uninfected cell population; Q4, GFP-negative (DC-SIGN-negative) and uninfected cell population; ( B , C ) Jurkat-DC-SIGN cells were infected with rMP-12 or that lacking one ( B ) or two sequons ( C ). Relative number of RVFV-infected cells in the GFP-positive cell population (Q2/(Q2+Q3)) normalized to that of RVFV-infected cells in the GFP-negative cell population (Q1/(Q1+Q4)) are shown. Graphs represent mean + standard deviations for three independent experiments.
    Anti Mouse Igg Conjugated To Alexa Fluor 647, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse igg1 af647
    Infectivity of recombinant MP-12 encoding a glutamine (Q) in place of an asparagine (N) at N-X-S/T sequon(s) in Gn or Gc. ( A ) Jurkat-DC-SIGN cells stably co-expressing green fluorescent protein (GFP) and human DC-SIGN (~17% in cell population) (top panels), or parental Jurkat cells, which express neither DC-SIGN nor GFP (bottom panels), were mock-infected (left and center panels) or infected with rMP-12 (right panels) at a multiplicity of infection (MOI) of 3.6. At 6 hpi, cells were fixed, permeabilized, and then stained with a cocktail of mouse anti-RVFV antibody and <t>Alexa</t> Fluor 488-conjugated rabbit anti-GFP antibody, or a cocktail of mouse anti-RVFV antibody and Alexa Fluor 488-conjugated normal rabbit <t>IgG.</t> Subsequently, cells were stained with Alexa Fluor 647-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. Since permeabilized Jurkat-DC-SIGN cells showed poor intrinsic GFP signals, as shown with Alexa Fluor 488-conjugated normal rabbit IgG (left top panel), rabbit anti-GFP antibody (center and right top panels) was used to detect GFP signals of permeabilized Jurkat-DC-SIGN cells. Q1, GFP-negative (DC-SIGN-negative) and RVFV-infected cell population; Q2, GFP-positive (DC-SIGN-positive) and RVFV-infected cell population; Q3, GFP-positive (DC-SIGN-positive) and uninfected cell population; Q4, GFP-negative (DC-SIGN-negative) and uninfected cell population; ( B , C ) Jurkat-DC-SIGN cells were infected with rMP-12 or that lacking one ( B ) or two sequons ( C ). Relative number of RVFV-infected cells in the GFP-positive cell population (Q2/(Q2+Q3)) normalized to that of RVFV-infected cells in the GFP-negative cell population (Q1/(Q1+Q4)) are shown. Graphs represent mean + standard deviations for three independent experiments.
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    Thermo Fisher goat anti mouse igg
    Infectivity of recombinant MP-12 encoding a glutamine (Q) in place of an asparagine (N) at N-X-S/T sequon(s) in Gn or Gc. ( A ) Jurkat-DC-SIGN cells stably co-expressing green fluorescent protein (GFP) and human DC-SIGN (~17% in cell population) (top panels), or parental Jurkat cells, which express neither DC-SIGN nor GFP (bottom panels), were mock-infected (left and center panels) or infected with rMP-12 (right panels) at a multiplicity of infection (MOI) of 3.6. At 6 hpi, cells were fixed, permeabilized, and then stained with a cocktail of mouse anti-RVFV antibody and <t>Alexa</t> Fluor 488-conjugated rabbit anti-GFP antibody, or a cocktail of mouse anti-RVFV antibody and Alexa Fluor 488-conjugated normal rabbit <t>IgG.</t> Subsequently, cells were stained with Alexa Fluor 647-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. Since permeabilized Jurkat-DC-SIGN cells showed poor intrinsic GFP signals, as shown with Alexa Fluor 488-conjugated normal rabbit IgG (left top panel), rabbit anti-GFP antibody (center and right top panels) was used to detect GFP signals of permeabilized Jurkat-DC-SIGN cells. Q1, GFP-negative (DC-SIGN-negative) and RVFV-infected cell population; Q2, GFP-positive (DC-SIGN-positive) and RVFV-infected cell population; Q3, GFP-positive (DC-SIGN-positive) and uninfected cell population; Q4, GFP-negative (DC-SIGN-negative) and uninfected cell population; ( B , C ) Jurkat-DC-SIGN cells were infected with rMP-12 or that lacking one ( B ) or two sequons ( C ). Relative number of RVFV-infected cells in the GFP-positive cell population (Q2/(Q2+Q3)) normalized to that of RVFV-infected cells in the GFP-negative cell population (Q1/(Q1+Q4)) are shown. Graphs represent mean + standard deviations for three independent experiments.
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    Infectivity of recombinant MP-12 encoding a glutamine (Q) in place of an asparagine (N) at N-X-S/T sequon(s) in Gn or Gc. ( A ) Jurkat-DC-SIGN cells stably co-expressing green fluorescent protein (GFP) and human DC-SIGN (~17% in cell population) (top panels), or parental Jurkat cells, which express neither DC-SIGN nor GFP (bottom panels), were mock-infected (left and center panels) or infected with rMP-12 (right panels) at a multiplicity of infection (MOI) of 3.6. At 6 hpi, cells were fixed, permeabilized, and then stained with a cocktail of mouse anti-RVFV antibody and Alexa Fluor 488-conjugated rabbit anti-GFP antibody, or a cocktail of mouse anti-RVFV antibody and Alexa Fluor 488-conjugated normal rabbit IgG. Subsequently, cells were stained with Alexa Fluor 647-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. Since permeabilized Jurkat-DC-SIGN cells showed poor intrinsic GFP signals, as shown with Alexa Fluor 488-conjugated normal rabbit IgG (left top panel), rabbit anti-GFP antibody (center and right top panels) was used to detect GFP signals of permeabilized Jurkat-DC-SIGN cells. Q1, GFP-negative (DC-SIGN-negative) and RVFV-infected cell population; Q2, GFP-positive (DC-SIGN-positive) and RVFV-infected cell population; Q3, GFP-positive (DC-SIGN-positive) and uninfected cell population; Q4, GFP-negative (DC-SIGN-negative) and uninfected cell population; ( B , C ) Jurkat-DC-SIGN cells were infected with rMP-12 or that lacking one ( B ) or two sequons ( C ). Relative number of RVFV-infected cells in the GFP-positive cell population (Q2/(Q2+Q3)) normalized to that of RVFV-infected cells in the GFP-negative cell population (Q1/(Q1+Q4)) are shown. Graphs represent mean + standard deviations for three independent experiments.

    Journal: Viruses

    Article Title: N-Glycans on the Rift Valley Fever Virus Envelope Glycoproteins Gn and Gc Redundantly Support Viral Infection via DC-SIGN

    doi: 10.3390/v8050149

    Figure Lengend Snippet: Infectivity of recombinant MP-12 encoding a glutamine (Q) in place of an asparagine (N) at N-X-S/T sequon(s) in Gn or Gc. ( A ) Jurkat-DC-SIGN cells stably co-expressing green fluorescent protein (GFP) and human DC-SIGN (~17% in cell population) (top panels), or parental Jurkat cells, which express neither DC-SIGN nor GFP (bottom panels), were mock-infected (left and center panels) or infected with rMP-12 (right panels) at a multiplicity of infection (MOI) of 3.6. At 6 hpi, cells were fixed, permeabilized, and then stained with a cocktail of mouse anti-RVFV antibody and Alexa Fluor 488-conjugated rabbit anti-GFP antibody, or a cocktail of mouse anti-RVFV antibody and Alexa Fluor 488-conjugated normal rabbit IgG. Subsequently, cells were stained with Alexa Fluor 647-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. Since permeabilized Jurkat-DC-SIGN cells showed poor intrinsic GFP signals, as shown with Alexa Fluor 488-conjugated normal rabbit IgG (left top panel), rabbit anti-GFP antibody (center and right top panels) was used to detect GFP signals of permeabilized Jurkat-DC-SIGN cells. Q1, GFP-negative (DC-SIGN-negative) and RVFV-infected cell population; Q2, GFP-positive (DC-SIGN-positive) and RVFV-infected cell population; Q3, GFP-positive (DC-SIGN-positive) and uninfected cell population; Q4, GFP-negative (DC-SIGN-negative) and uninfected cell population; ( B , C ) Jurkat-DC-SIGN cells were infected with rMP-12 or that lacking one ( B ) or two sequons ( C ). Relative number of RVFV-infected cells in the GFP-positive cell population (Q2/(Q2+Q3)) normalized to that of RVFV-infected cells in the GFP-negative cell population (Q1/(Q1+Q4)) are shown. Graphs represent mean + standard deviations for three independent experiments.

    Article Snippet: Cells were washed twice with permeabilization buffer, and the cells were incubated at 4 °C for 40 min with Alexa Fluor 647-conjugated goat anti-mouse IgG (Life Technologies).

    Techniques: Infection, Recombinant, Stable Transfection, Expressing, Staining, Flow Cytometry, Cytometry

    Staurosporine treatment results in the redistribution of SSA/Ro52 to surface blebs of apoptotic cells. A representative confocal microcopy image of RAW264.7 cells treated with 1 µM staurosporine for 6 h. The distribution of the SSA/Ro52 autoantigen was visualized through confocal microscopy using a rabbit polyclonal anti-SSA/Ro52 and an Alexa Fluor 488 conjugated goat anti-rabbit IgG secondary (Panel A). Nuclei were counterstained with propidium iodide (Panel B). Apoptotic nuclei are indicated by arrows. The plasma membrane was visualized with Alexa Fluor 647 cholera toxin subunit B conjugate (Panel C). Panel D shows the merged image with an arrow indicating apoptotic cell surface blebs enriched in SSA/Ro52.

    Journal: Toxicology

    Article Title: Autoantibodies from mice exposed to Libby amphibole asbestos bind SSA/Ro52 - enriched apoptotic blebs of murine macrophages

    doi: 10.1016/j.tox.2008.01.008

    Figure Lengend Snippet: Staurosporine treatment results in the redistribution of SSA/Ro52 to surface blebs of apoptotic cells. A representative confocal microcopy image of RAW264.7 cells treated with 1 µM staurosporine for 6 h. The distribution of the SSA/Ro52 autoantigen was visualized through confocal microscopy using a rabbit polyclonal anti-SSA/Ro52 and an Alexa Fluor 488 conjugated goat anti-rabbit IgG secondary (Panel A). Nuclei were counterstained with propidium iodide (Panel B). Apoptotic nuclei are indicated by arrows. The plasma membrane was visualized with Alexa Fluor 647 cholera toxin subunit B conjugate (Panel C). Panel D shows the merged image with an arrow indicating apoptotic cell surface blebs enriched in SSA/Ro52.

    Article Snippet: Bound autoantibodies from asbestos exposed mice were detected using an Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Molecular Probes).

    Techniques: Confocal Microscopy

    Libby 6-mix exposure results in the redistribution of SSA/Ro52 to surface blebs of apoptotic cells. Representative confocal microcopy images of RAW264.7 cells untreated (Panels A–D) or exposed to Libby asbestos for 48 h (Panels E–L). The distribution of the SSA/Ro52 autoantigen was visualized through confocal microscopy using a rabbit polyclonal anti-SSA/Ro52 and an Alexa Fluor 488 conjugated goat anti-rabbit IgG secondary (Green in panels A, E and I). Nuclei were counterstained with propidium iodide (Red in panels B, F and J). Apoptotic nuclei are indicated by arrows. The plasma membrane was visualized with Alexa Fluor 647 cholera toxin subunit B conjugate (Magenta in panels C, G and K). Right panels (D, H and L) show merged images with arrows indicating apoptotic cell surface blebs enriched in SSA/Ro52.

    Journal: Toxicology

    Article Title: Autoantibodies from mice exposed to Libby amphibole asbestos bind SSA/Ro52 - enriched apoptotic blebs of murine macrophages

    doi: 10.1016/j.tox.2008.01.008

    Figure Lengend Snippet: Libby 6-mix exposure results in the redistribution of SSA/Ro52 to surface blebs of apoptotic cells. Representative confocal microcopy images of RAW264.7 cells untreated (Panels A–D) or exposed to Libby asbestos for 48 h (Panels E–L). The distribution of the SSA/Ro52 autoantigen was visualized through confocal microscopy using a rabbit polyclonal anti-SSA/Ro52 and an Alexa Fluor 488 conjugated goat anti-rabbit IgG secondary (Green in panels A, E and I). Nuclei were counterstained with propidium iodide (Red in panels B, F and J). Apoptotic nuclei are indicated by arrows. The plasma membrane was visualized with Alexa Fluor 647 cholera toxin subunit B conjugate (Magenta in panels C, G and K). Right panels (D, H and L) show merged images with arrows indicating apoptotic cell surface blebs enriched in SSA/Ro52.

    Article Snippet: Bound autoantibodies from asbestos exposed mice were detected using an Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Molecular Probes).

    Techniques: Confocal Microscopy

    Autoantibodies from asbestos-exposed mice recognize apoptotic blebs enriched with the SSA/Ro52 autoantigen. Representative confocal microcopy images of RAW264.7 cells exposed to Libby asbestos for 48 h. The SSA/Ro52 autoantigen was visualized through confocal microscopy using a rabbit polyclonal anti-SSA/Ro52 antibody and an Alexa Fluor 488 conjugated goat anti-rabbit IgG secondary (Green in panels A, E and I). Nuclei were counterstained with propidium iodide (Cyan in panels B, F and J). Apoptotic nuclei are indicated by arrows. Binding of mouse autoantibodies were visualized with an Alexa Fluor 647 conjugated goat anti-mouse IgG secondary (Red in panels C, G and K). Panels C and G include AA staining from a mouse exposed to tremolite asbestos. Panel K includes AA staining from a mouse exposed to wollastonite fibers. Right panels (D, H and L) show merged images. Colocalization of SSA/Ro52 and autoantigens recognized by asbestos-induced mouse AAs is visualized in yellow in merged images and indicated by arrows. Colocalization was confirmed through Image J analysis.

    Journal: Toxicology

    Article Title: Autoantibodies from mice exposed to Libby amphibole asbestos bind SSA/Ro52 - enriched apoptotic blebs of murine macrophages

    doi: 10.1016/j.tox.2008.01.008

    Figure Lengend Snippet: Autoantibodies from asbestos-exposed mice recognize apoptotic blebs enriched with the SSA/Ro52 autoantigen. Representative confocal microcopy images of RAW264.7 cells exposed to Libby asbestos for 48 h. The SSA/Ro52 autoantigen was visualized through confocal microscopy using a rabbit polyclonal anti-SSA/Ro52 antibody and an Alexa Fluor 488 conjugated goat anti-rabbit IgG secondary (Green in panels A, E and I). Nuclei were counterstained with propidium iodide (Cyan in panels B, F and J). Apoptotic nuclei are indicated by arrows. Binding of mouse autoantibodies were visualized with an Alexa Fluor 647 conjugated goat anti-mouse IgG secondary (Red in panels C, G and K). Panels C and G include AA staining from a mouse exposed to tremolite asbestos. Panel K includes AA staining from a mouse exposed to wollastonite fibers. Right panels (D, H and L) show merged images. Colocalization of SSA/Ro52 and autoantigens recognized by asbestos-induced mouse AAs is visualized in yellow in merged images and indicated by arrows. Colocalization was confirmed through Image J analysis.

    Article Snippet: Bound autoantibodies from asbestos exposed mice were detected using an Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Molecular Probes).

    Techniques: Mouse Assay, Confocal Microscopy, Binding Assay, Staining, Atomic Absorption Spectroscopy

    HASPB18–GFP is exposed on the surface of live metacyclic L. major . A. Surface HASPB18–GFP exposure was determined by FACS analysis of live HASPB18–GFP L. major (following labelling with Sulfo-NHS-AMCA to distinguish between live and dead cells) using mouse anti-GFP and detection by AlexaFluor-647-conjugated goat anti-mouse IgG. Non-N-myristoylated HASPB18–GFP G2A parasites were used as the control for non-surface exposure. The live/dead analysis (left hand panel) detected 2% dead cells in the HASPB18–GFP parasite population; the remaining analyses (centre and right hand panels) are gated on live cells only. EC GFP, extracellular GFP. B. Confocal microscopic analysis of a metacyclic HASPB18–GFP L. major labelled using the protocol in (A); extracellular HASPB18–GFP, detected by mouse anti-GFP, decorating the surface of the cell body and flagellum in a punctate distribution, while intracellular protein was detected by GFP fluorescence. Size bar, 5 µm. C. Scanning immunoelectron microscopy of a metacyclic HASPB18–GFP L. major incubated live with mouse anti-GFP, prior to fixation in 4% paraformaldehyde and incubation with goat anti-mouse IgG 10 nm gold. Samples were post-fixed in 2.5% glutaraldehyde, dehydrated and carbon coated, prior to visualization. White arrows indicate surface-localized gold labelling, F indicates flagellum protruding from parasite body. D. Transmission immunoelectron microscopy of HASPB18–GFP L. major parasites, labelled as in (C). Samples were post-fixed in 2.5% glutaraldehyde/4% formaldehyde and resin embedded, prior to sectioning and visualization. The black box within the whole-cell image represents the area enlarged on the right. Black arrow indicates gold labelling on a surface vesicle.

    Journal: Cellular Microbiology

    Article Title: Trafficking and release of Leishmania metacyclic HASPB on macrophage invasion

    doi: 10.1111/j.1462-5822.2012.01756.x

    Figure Lengend Snippet: HASPB18–GFP is exposed on the surface of live metacyclic L. major . A. Surface HASPB18–GFP exposure was determined by FACS analysis of live HASPB18–GFP L. major (following labelling with Sulfo-NHS-AMCA to distinguish between live and dead cells) using mouse anti-GFP and detection by AlexaFluor-647-conjugated goat anti-mouse IgG. Non-N-myristoylated HASPB18–GFP G2A parasites were used as the control for non-surface exposure. The live/dead analysis (left hand panel) detected 2% dead cells in the HASPB18–GFP parasite population; the remaining analyses (centre and right hand panels) are gated on live cells only. EC GFP, extracellular GFP. B. Confocal microscopic analysis of a metacyclic HASPB18–GFP L. major labelled using the protocol in (A); extracellular HASPB18–GFP, detected by mouse anti-GFP, decorating the surface of the cell body and flagellum in a punctate distribution, while intracellular protein was detected by GFP fluorescence. Size bar, 5 µm. C. Scanning immunoelectron microscopy of a metacyclic HASPB18–GFP L. major incubated live with mouse anti-GFP, prior to fixation in 4% paraformaldehyde and incubation with goat anti-mouse IgG 10 nm gold. Samples were post-fixed in 2.5% glutaraldehyde, dehydrated and carbon coated, prior to visualization. White arrows indicate surface-localized gold labelling, F indicates flagellum protruding from parasite body. D. Transmission immunoelectron microscopy of HASPB18–GFP L. major parasites, labelled as in (C). Samples were post-fixed in 2.5% glutaraldehyde/4% formaldehyde and resin embedded, prior to sectioning and visualization. The black box within the whole-cell image represents the area enlarged on the right. Black arrow indicates gold labelling on a surface vesicle.

    Article Snippet: Cells were then washed three times with cold 1% fatty acid-free BSA blocking solution (BB International) and resuspended in 100 µl of blocking solution for 20 min. To detect extracellular (EC) HASPB and lipophosphoglycan (LPG), parasites were labelled with rabbit anti-HASPB336 (1:300) ( ) and/or mouse 3F12 (undiluted) ( ) for 30 min at 20°C, then fixed in 4% paraformaldehyde (PFA) before secondary detection with AlexaFluor-488-conjugated goat anti-rabbit IgG and AlexaFluor-647-conjugated goat anti-mouse IgG respectively (1:250 in blocking solution, Invitrogen).

    Techniques: FACS, Fluorescence, Immuno-Electron Microscopy, Incubation, Transmission Assay

    Full-length HASPB is exposed on the surface of metacyclic L. major . A and B. Flow cytometry analysis of early passage (p = 3) and late passage (p > 10) L. major FVI wild-type parasites sampled from early log phase (Day 2), late log phase (Day 5) and stationary, metacyclic-rich phase (Day 7). Sulfo-NHS-AMCA staining was used to distinguish live/dead cells to allow gating on live parasites only, as in Fig. 4 . Live parasites were labelled for detection of EC HASPB (using polyclonal antibody 336 and AlexaFluor-488-conjugated secondary antibody) and metacyclic-specific LPG (EC LPG 3F12, using monoclonal 3F12 antibody and AlexaFluor-647-conjugated secondary antibody). C. Parasites from the same early and late passage cultures were lysed and analysed for HASPB expression by immunoblotting, using EF1-α as a control for equivalent loading. HASPB migrates as a ∼ 36 kDa band under these conditions while EF1-α recognizes a 50 kDa band as the major protein. D. Confocal microscopy of live anti-HASPB- and 3F12-labelled early passage L. major (as described in A and B) confirmed surface exposure of full-length HASPB across the entire cell body and flagellum in a punctate pattern. The DAPI-stained kinetoplast and nucleus are visible and their relative positions identify this cell as a metacyclic parasite; this identification is also verified by staining with the metacyclic-specific antibody, 3F12. Extracellular (EC) HASPB colocalized with EC metacyclic LPG in this parasite, which was intact at the time of labelling as indicated by the absence of Sulfo-NHS-AMCA staining. Size bar, 5 µm. E and F. (E) Scanning immunoelectron microscopy and (F) transmission immunoelectron microscopy of live anti-HASPB-labelled early passage stationary-phase L. major detected with goat anti-rabbit IgG 10 nm gold. White arrows indicate gold particles in SEM image. The black box within the whole-cell image TIEM represents the area enlarged on the right. Black arrows indicate labelled surface vesicles. F, flagellum. G. HASPB expression and shedding by early passage wild-type L. major and LmcDNA16 mutant metacyclic parasites either null for (K0) or re-expressing (KI) the LmcDNA16 locus. Cell fractionation of Day 7 cultures was used to generate membrane (Mb) and soluble (Sol) fractions, while supernatant fractions (S/N) were collected prior to parasite lysis. HASPB levels were determined by immunoblotting with anti-HASPB 336 or anti-EF1-α as in (C).

    Journal: Cellular Microbiology

    Article Title: Trafficking and release of Leishmania metacyclic HASPB on macrophage invasion

    doi: 10.1111/j.1462-5822.2012.01756.x

    Figure Lengend Snippet: Full-length HASPB is exposed on the surface of metacyclic L. major . A and B. Flow cytometry analysis of early passage (p = 3) and late passage (p > 10) L. major FVI wild-type parasites sampled from early log phase (Day 2), late log phase (Day 5) and stationary, metacyclic-rich phase (Day 7). Sulfo-NHS-AMCA staining was used to distinguish live/dead cells to allow gating on live parasites only, as in Fig. 4 . Live parasites were labelled for detection of EC HASPB (using polyclonal antibody 336 and AlexaFluor-488-conjugated secondary antibody) and metacyclic-specific LPG (EC LPG 3F12, using monoclonal 3F12 antibody and AlexaFluor-647-conjugated secondary antibody). C. Parasites from the same early and late passage cultures were lysed and analysed for HASPB expression by immunoblotting, using EF1-α as a control for equivalent loading. HASPB migrates as a ∼ 36 kDa band under these conditions while EF1-α recognizes a 50 kDa band as the major protein. D. Confocal microscopy of live anti-HASPB- and 3F12-labelled early passage L. major (as described in A and B) confirmed surface exposure of full-length HASPB across the entire cell body and flagellum in a punctate pattern. The DAPI-stained kinetoplast and nucleus are visible and their relative positions identify this cell as a metacyclic parasite; this identification is also verified by staining with the metacyclic-specific antibody, 3F12. Extracellular (EC) HASPB colocalized with EC metacyclic LPG in this parasite, which was intact at the time of labelling as indicated by the absence of Sulfo-NHS-AMCA staining. Size bar, 5 µm. E and F. (E) Scanning immunoelectron microscopy and (F) transmission immunoelectron microscopy of live anti-HASPB-labelled early passage stationary-phase L. major detected with goat anti-rabbit IgG 10 nm gold. White arrows indicate gold particles in SEM image. The black box within the whole-cell image TIEM represents the area enlarged on the right. Black arrows indicate labelled surface vesicles. F, flagellum. G. HASPB expression and shedding by early passage wild-type L. major and LmcDNA16 mutant metacyclic parasites either null for (K0) or re-expressing (KI) the LmcDNA16 locus. Cell fractionation of Day 7 cultures was used to generate membrane (Mb) and soluble (Sol) fractions, while supernatant fractions (S/N) were collected prior to parasite lysis. HASPB levels were determined by immunoblotting with anti-HASPB 336 or anti-EF1-α as in (C).

    Article Snippet: Cells were then washed three times with cold 1% fatty acid-free BSA blocking solution (BB International) and resuspended in 100 µl of blocking solution for 20 min. To detect extracellular (EC) HASPB and lipophosphoglycan (LPG), parasites were labelled with rabbit anti-HASPB336 (1:300) ( ) and/or mouse 3F12 (undiluted) ( ) for 30 min at 20°C, then fixed in 4% paraformaldehyde (PFA) before secondary detection with AlexaFluor-488-conjugated goat anti-rabbit IgG and AlexaFluor-647-conjugated goat anti-mouse IgG respectively (1:250 in blocking solution, Invitrogen).

    Techniques: Flow Cytometry, Cytometry, Staining, Expressing, Confocal Microscopy, Immuno-Electron Microscopy, Transmission Assay, Mutagenesis, Cell Fractionation, Lysis

    HASPB18–GFP localization in L. major promastigotes. Both fixed and live cell imaging methods were used to localize the HASPB18–GFP (A) and HASPB18–GFP C3S fusion proteins (B) in transfected L. major parasites [all parasites analysed were early passage cells (≤ p6)]. A and B. Top panel: Metacyclic parasites expressing HASPB18–GFP or HASPB18–GFP C3S were fixed and permeabilized, prior to labelling with rabbit anti-YPT and subsequent detection using AlexaFluor-647-conjugated goat anti-rabbit IgG. Lower three panels: Live metacyclics or procyclics expressing either GFP fusion protein were imaged after immobilization in PBS-primed CyGEL and labelling with Lysotracker RED DND-99 or FM4-64 for 90 min. *Vesicular structures of unknown origin. C and D. Live cell staining of HASPB18–GFP (C) and C3S (D) parasites that are also expressing the autophagosomal marker, RFP-ATG8. E. Density gradient fractionation of cell lysates from metacyclic parasites of the HASPB18–GFP/RFP-ATG8 lines analysed in (C) and (D), immunoblotted with anti-GFP, anti-RFP or anti-GP63. Arrowheads indicate subcellular compartments as follows: G, Golgi; L, lysosome; FP, flagellar pocket; E, endosome; V, vesicle; AP, autophagosome. Size bar, 5 µm (all images at the same magnification).

    Journal: Cellular Microbiology

    Article Title: Trafficking and release of Leishmania metacyclic HASPB on macrophage invasion

    doi: 10.1111/j.1462-5822.2012.01756.x

    Figure Lengend Snippet: HASPB18–GFP localization in L. major promastigotes. Both fixed and live cell imaging methods were used to localize the HASPB18–GFP (A) and HASPB18–GFP C3S fusion proteins (B) in transfected L. major parasites [all parasites analysed were early passage cells (≤ p6)]. A and B. Top panel: Metacyclic parasites expressing HASPB18–GFP or HASPB18–GFP C3S were fixed and permeabilized, prior to labelling with rabbit anti-YPT and subsequent detection using AlexaFluor-647-conjugated goat anti-rabbit IgG. Lower three panels: Live metacyclics or procyclics expressing either GFP fusion protein were imaged after immobilization in PBS-primed CyGEL and labelling with Lysotracker RED DND-99 or FM4-64 for 90 min. *Vesicular structures of unknown origin. C and D. Live cell staining of HASPB18–GFP (C) and C3S (D) parasites that are also expressing the autophagosomal marker, RFP-ATG8. E. Density gradient fractionation of cell lysates from metacyclic parasites of the HASPB18–GFP/RFP-ATG8 lines analysed in (C) and (D), immunoblotted with anti-GFP, anti-RFP or anti-GP63. Arrowheads indicate subcellular compartments as follows: G, Golgi; L, lysosome; FP, flagellar pocket; E, endosome; V, vesicle; AP, autophagosome. Size bar, 5 µm (all images at the same magnification).

    Article Snippet: Cells were then washed three times with cold 1% fatty acid-free BSA blocking solution (BB International) and resuspended in 100 µl of blocking solution for 20 min. To detect extracellular (EC) HASPB and lipophosphoglycan (LPG), parasites were labelled with rabbit anti-HASPB336 (1:300) ( ) and/or mouse 3F12 (undiluted) ( ) for 30 min at 20°C, then fixed in 4% paraformaldehyde (PFA) before secondary detection with AlexaFluor-488-conjugated goat anti-rabbit IgG and AlexaFluor-647-conjugated goat anti-mouse IgG respectively (1:250 in blocking solution, Invitrogen).

    Techniques: Live Cell Imaging, Transfection, Expressing, Staining, Marker, Fractionation

    HIV-1 Gag failed to localize to VCCs upon 5ptaseIV overexpression. (A) MDMs were infected with pseudotyped HIV-1 encoding Gag-YFP along with 5ptaseIV (FL) or 5ptaseIV Δ1. At 48 h postinfection, cells were stained with ConA labeled with Alexa Fluor 594 (a PM marker), fixed, immunostained with a mouse monoclonal anti-CD81 antibody and anti-mouse IgG conjugated with Alexa Fluor 647, and analyzed using a confocal microscope. (B) Pearson's correlation coefficients for colocalization of Gag-YFP with CD81 are shown as means and standard errors of the means (SEM). Twenty to 30 cells were analyzed per condition. **, P

    Journal: Journal of Virology

    Article Title: Molecular Determinants Directing HIV-1 Gag Assembly to Virus-Containing Compartments in Primary Macrophages

    doi: 10.1128/JVI.01004-16

    Figure Lengend Snippet: HIV-1 Gag failed to localize to VCCs upon 5ptaseIV overexpression. (A) MDMs were infected with pseudotyped HIV-1 encoding Gag-YFP along with 5ptaseIV (FL) or 5ptaseIV Δ1. At 48 h postinfection, cells were stained with ConA labeled with Alexa Fluor 594 (a PM marker), fixed, immunostained with a mouse monoclonal anti-CD81 antibody and anti-mouse IgG conjugated with Alexa Fluor 647, and analyzed using a confocal microscope. (B) Pearson's correlation coefficients for colocalization of Gag-YFP with CD81 are shown as means and standard errors of the means (SEM). Twenty to 30 cells were analyzed per condition. **, P

    Article Snippet: The cells were immunostained with anti-CD81 antibody (BD Biosciences Pharmingen, San Diego, CA) for 1 h at room temperature, washed twice with PBS, and stained with goat anti-mouse IgG conjugated to Alexa Fluor 647 (Invitrogen).

    Techniques: Over Expression, Infection, Staining, Labeling, Marker, Microscopy

    The myristate moiety but not the intact HIV-1 MA sequence is required for VCC localization. (A) MDMs were infected with pseudotyped HIV-1 encoding WT Gag-YFP or Gag-YFP derivatives containing the indicated MA substitutions. At 48 h postinfection, cells were stained with ConA labeled with Alexa Fluor 594, fixed, immunostained with a mouse monoclonal anti-CD81 antibody and anti-mouse IgG conjugated with Alexa Fluor 647, and analyzed using a confocal microscope. (B) Pearson's correlation coefficients for colocalization of Gag-YFP with CD81 are shown as means and SEM. Twenty to 30 cells were analyzed per condition. *, P

    Journal: Journal of Virology

    Article Title: Molecular Determinants Directing HIV-1 Gag Assembly to Virus-Containing Compartments in Primary Macrophages

    doi: 10.1128/JVI.01004-16

    Figure Lengend Snippet: The myristate moiety but not the intact HIV-1 MA sequence is required for VCC localization. (A) MDMs were infected with pseudotyped HIV-1 encoding WT Gag-YFP or Gag-YFP derivatives containing the indicated MA substitutions. At 48 h postinfection, cells were stained with ConA labeled with Alexa Fluor 594, fixed, immunostained with a mouse monoclonal anti-CD81 antibody and anti-mouse IgG conjugated with Alexa Fluor 647, and analyzed using a confocal microscope. (B) Pearson's correlation coefficients for colocalization of Gag-YFP with CD81 are shown as means and SEM. Twenty to 30 cells were analyzed per condition. *, P

    Article Snippet: The cells were immunostained with anti-CD81 antibody (BD Biosciences Pharmingen, San Diego, CA) for 1 h at room temperature, washed twice with PBS, and stained with goat anti-mouse IgG conjugated to Alexa Fluor 647 (Invitrogen).

    Techniques: Sequencing, Infection, Staining, Labeling, Microscopy

    Heterologous membrane binding sequences can replace HIV-1 MA without affecting VCC localization. (A) Schematic illustrations of WT, PH/ΔMA, Kmyr/ΔMA, and Fyn(10)/ΔMA Gag-YFP. (B) MDMs were infected with pseudotyped HIV-1 encoding WT Gag-YFP or Gag-YFP derivatives in which MA was replaced by a heterologous membrane binding sequence. At 48 h postinfection, cells were stained with ConA labeled with Alexa Fluor 594, fixed, immunostained with a mouse monoclonal anti-CD81 antibody and anti-mouse IgG conjugated with Alexa Fluor 647, and analyzed using a confocal microscope. (C) Pearson's correlation coefficients for colocalization of Gag-YFP with CD81 are shown as means and SEM. Twenty to 30 cells were analyzed per condition. n.s., not significant.

    Journal: Journal of Virology

    Article Title: Molecular Determinants Directing HIV-1 Gag Assembly to Virus-Containing Compartments in Primary Macrophages

    doi: 10.1128/JVI.01004-16

    Figure Lengend Snippet: Heterologous membrane binding sequences can replace HIV-1 MA without affecting VCC localization. (A) Schematic illustrations of WT, PH/ΔMA, Kmyr/ΔMA, and Fyn(10)/ΔMA Gag-YFP. (B) MDMs were infected with pseudotyped HIV-1 encoding WT Gag-YFP or Gag-YFP derivatives in which MA was replaced by a heterologous membrane binding sequence. At 48 h postinfection, cells were stained with ConA labeled with Alexa Fluor 594, fixed, immunostained with a mouse monoclonal anti-CD81 antibody and anti-mouse IgG conjugated with Alexa Fluor 647, and analyzed using a confocal microscope. (C) Pearson's correlation coefficients for colocalization of Gag-YFP with CD81 are shown as means and SEM. Twenty to 30 cells were analyzed per condition. n.s., not significant.

    Article Snippet: The cells were immunostained with anti-CD81 antibody (BD Biosciences Pharmingen, San Diego, CA) for 1 h at room temperature, washed twice with PBS, and stained with goat anti-mouse IgG conjugated to Alexa Fluor 647 (Invitrogen).

    Techniques: Binding Assay, Infection, Sequencing, Staining, Labeling, Microscopy

    HIV-1 Gag localization to VCCs requires higher-order multimerization. (A) Schematic illustrations of WT, EE75,76AA, delNC, LZ, and LZ4 Gag-YFP. LZ4 Gag-YFP contains the WM184,185AA CA mutation, which disrupts Gag dimerization, in addition to replacement of NC with a tetramer-forming LZ sequence. (B) MDMs were infected with pseudotyped HIV-1 encoding WT Gag-YFP or Gag-YFP derivatives containing the substitutions shown in panel A. At 48 h postinfection, cells were stained with ConA labeled with Alexa Fluor 594, fixed, immunostained with a mouse monoclonal anti-CD81 antibody and anti-mouse IgG conjugated with Alexa Fluor 647, and analyzed using a confocal microscope. Note that for cells expressing delNC Gag-YFP or LZ4 Gag-YFP, prominent YFP signals were found at the cell surface as detected by ConA (white arrowheads), whereas such surface YFP signals were nearly undetectable in WT Gag-YFP-expressing cells. (C) Pearson's correlation coefficients for colocalization of Gag-YFP with ConA are shown as means and SEM. Twenty to 40 cells were analyzed per condition. *, P

    Journal: Journal of Virology

    Article Title: Molecular Determinants Directing HIV-1 Gag Assembly to Virus-Containing Compartments in Primary Macrophages

    doi: 10.1128/JVI.01004-16

    Figure Lengend Snippet: HIV-1 Gag localization to VCCs requires higher-order multimerization. (A) Schematic illustrations of WT, EE75,76AA, delNC, LZ, and LZ4 Gag-YFP. LZ4 Gag-YFP contains the WM184,185AA CA mutation, which disrupts Gag dimerization, in addition to replacement of NC with a tetramer-forming LZ sequence. (B) MDMs were infected with pseudotyped HIV-1 encoding WT Gag-YFP or Gag-YFP derivatives containing the substitutions shown in panel A. At 48 h postinfection, cells were stained with ConA labeled with Alexa Fluor 594, fixed, immunostained with a mouse monoclonal anti-CD81 antibody and anti-mouse IgG conjugated with Alexa Fluor 647, and analyzed using a confocal microscope. Note that for cells expressing delNC Gag-YFP or LZ4 Gag-YFP, prominent YFP signals were found at the cell surface as detected by ConA (white arrowheads), whereas such surface YFP signals were nearly undetectable in WT Gag-YFP-expressing cells. (C) Pearson's correlation coefficients for colocalization of Gag-YFP with ConA are shown as means and SEM. Twenty to 40 cells were analyzed per condition. *, P

    Article Snippet: The cells were immunostained with anti-CD81 antibody (BD Biosciences Pharmingen, San Diego, CA) for 1 h at room temperature, washed twice with PBS, and stained with goat anti-mouse IgG conjugated to Alexa Fluor 647 (Invitrogen).

    Techniques: Mutagenesis, Sequencing, Infection, Staining, Labeling, Microscopy, Expressing