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anti‐ifnar1 neutralizing antibody  (Bio X Cell)


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    Bio X Cell anti‐ifnar1 neutralizing antibody
    Type I interferon signaling is critical for Mn‐N/C‐mediated anti‐tumor immune response. A) FACS analysis (left) of CFSE and the quantification (right) of OT‐1 T cell proliferation after co‐cultured with DCs sorted from lymph nodes of WT and <t>Ifnar1</t> −/− MC38‐OVA tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 4 per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. B) FACS analysis (below) and the quantification (above) of IFNγ + , Granzyme B + , TNFα + of OT‐1 CD8 + T cells after co‐culture with DCs as A described. Data are shown as mean±SEM ( n = 4 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. C) FACS analysis (above) and the quantification (below) of tumor‐infiltrating CD8 + T cells in WT and Ifnar1 −/− MC38 tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 5–6 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. D) FACS analysis (right) and the quantification (left) of IFNγ + , PRF1 + (Perforin), TNFα + of CD8 + T cells in WT and Ifnar1 −/− MC38 tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 5–6 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. E) Tumor growth curve of MC38 in WT and Ifnar1 −/− mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 5–6 mice per group). Two‐way ANOVA (mixed model) and Sidak's multiple comparisons test were performed. F) MC38 tumor weight in WT and Ifnar1 −/− mice with vehicle or Mn‐N/C treatment 14 days after tumor cell inoculation. Data are shown as mean±SEM ( n = 5–6 mice per group). G) Kaplan–Meier analysis of WT and Ifnar1 −/− mice survival with vehicle or Mn‐N/C treatment ( n = 5–6 mice per group). * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; ns, no significance
    Anti‐Ifnar1 Neutralizing Antibody, supplied by Bio X Cell, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti‐ifnar1 neutralizing antibody/product/Bio X Cell
    Average 90 stars, based on 1 article reviews
    anti‐ifnar1 neutralizing antibody - by Bioz Stars, 2026-03
    90/100 stars

    Images

    1) Product Images from "A Single‐Atom Manganese Nanozyme Mn‐N/C Promotes Anti‐Tumor Immune Response via Eliciting Type I Interferon Signaling"

    Article Title: A Single‐Atom Manganese Nanozyme Mn‐N/C Promotes Anti‐Tumor Immune Response via Eliciting Type I Interferon Signaling

    Journal: Advanced Science

    doi: 10.1002/advs.202305979

    Type I interferon signaling is critical for Mn‐N/C‐mediated anti‐tumor immune response. A) FACS analysis (left) of CFSE and the quantification (right) of OT‐1 T cell proliferation after co‐cultured with DCs sorted from lymph nodes of WT and Ifnar1 −/− MC38‐OVA tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 4 per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. B) FACS analysis (below) and the quantification (above) of IFNγ + , Granzyme B + , TNFα + of OT‐1 CD8 + T cells after co‐culture with DCs as A described. Data are shown as mean±SEM ( n = 4 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. C) FACS analysis (above) and the quantification (below) of tumor‐infiltrating CD8 + T cells in WT and Ifnar1 −/− MC38 tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 5–6 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. D) FACS analysis (right) and the quantification (left) of IFNγ + , PRF1 + (Perforin), TNFα + of CD8 + T cells in WT and Ifnar1 −/− MC38 tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 5–6 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. E) Tumor growth curve of MC38 in WT and Ifnar1 −/− mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 5–6 mice per group). Two‐way ANOVA (mixed model) and Sidak's multiple comparisons test were performed. F) MC38 tumor weight in WT and Ifnar1 −/− mice with vehicle or Mn‐N/C treatment 14 days after tumor cell inoculation. Data are shown as mean±SEM ( n = 5–6 mice per group). G) Kaplan–Meier analysis of WT and Ifnar1 −/− mice survival with vehicle or Mn‐N/C treatment ( n = 5–6 mice per group). * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; ns, no significance
    Figure Legend Snippet: Type I interferon signaling is critical for Mn‐N/C‐mediated anti‐tumor immune response. A) FACS analysis (left) of CFSE and the quantification (right) of OT‐1 T cell proliferation after co‐cultured with DCs sorted from lymph nodes of WT and Ifnar1 −/− MC38‐OVA tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 4 per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. B) FACS analysis (below) and the quantification (above) of IFNγ + , Granzyme B + , TNFα + of OT‐1 CD8 + T cells after co‐culture with DCs as A described. Data are shown as mean±SEM ( n = 4 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. C) FACS analysis (above) and the quantification (below) of tumor‐infiltrating CD8 + T cells in WT and Ifnar1 −/− MC38 tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 5–6 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. D) FACS analysis (right) and the quantification (left) of IFNγ + , PRF1 + (Perforin), TNFα + of CD8 + T cells in WT and Ifnar1 −/− MC38 tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 5–6 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. E) Tumor growth curve of MC38 in WT and Ifnar1 −/− mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 5–6 mice per group). Two‐way ANOVA (mixed model) and Sidak's multiple comparisons test were performed. F) MC38 tumor weight in WT and Ifnar1 −/− mice with vehicle or Mn‐N/C treatment 14 days after tumor cell inoculation. Data are shown as mean±SEM ( n = 5–6 mice per group). G) Kaplan–Meier analysis of WT and Ifnar1 −/− mice survival with vehicle or Mn‐N/C treatment ( n = 5–6 mice per group). * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; ns, no significance

    Techniques Used: Cell Culture, Co-Culture Assay

    A combination of Mn‐N/C and PD‐L1 blockade synergistically suppresses tumor growth. A) Real‐time PCR analysis of PD‐L1 mRNA expression of MC38 tumor cells treated with H 2 O 2 , Mn‐N/C for 24 h. Quantitative data are shown as mean±SEM ( n = 3). One‐way ANOVA and Sidak's multiple comparisons test were performed. B) FACS analysis (left) and the quantification (right) of PD‐L1 levels on the surface of MC38 tumor cells treated with H 2 O 2 , Mn‐N/C, or plus with anti‐IFNAR1 antibody or NAC treatment for 24 h. Quantitative data are shown as mean±SEM ( n = 3). One‐way ANOVA and Sidak's multiple comparisons test were performed. C) Real‐time PCR analysis of PD‐L1 mRNA expression of tumor cells (RFP + ) sorted from MC38‐OVA tumor‐bearing mice with vehicle or Mn‐N/C treatment. Quantitative data are shown as mean±SEM ( n = 3). Two‐tailed unpaired t ‐test was performed for the comparisons between the two groups. D) FACS analysis (left) and the quantification (right) of PD‐L1 levels in CD45 − cells from MC38 tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 6 mice per group). Two‐tailed unpaired t ‐test was performed for the comparisons between the two groups. E) Schematic illustration of a bilateral model of MC38 tumors subcutaneously implanted on C57BL/6 mice and treated with vehicle or Mn‐N/C, or plus with anti‐PD‐L1 antibody. F,G) Tumor growth curve of the first tumor (F) and the second tumor (G) of MC38 in the WT mice treated as E described. Data are shown as mean±SEM ( n = 5 mice per group). Two‐way ANOVA (mixed model) and Sidak's multiple comparisons test were performed. H,I) FACS analysis (left) and the quantification (right) of tumor‐infiltrating CD8 + T cells in the first tumor (H) and the second tumor (I) of MC38 tumor‐bearing mice treated as E described. Data are shown as mean±SEM ( n = 5 mice per group). Two‐tailed unpaired t ‐test was performed for the comparisons between the two groups. J,K) FACS analysis (left) and the quantification (right) of Granzyme B + , PRF1 + , TNFα + of CD8 + T cells in the first tumor (J) and the second tumor (K) of MC38 tumor‐bearing mice treated as E described. Data are shown as mean±SEM ( n = 5 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; ns, no significance
    Figure Legend Snippet: A combination of Mn‐N/C and PD‐L1 blockade synergistically suppresses tumor growth. A) Real‐time PCR analysis of PD‐L1 mRNA expression of MC38 tumor cells treated with H 2 O 2 , Mn‐N/C for 24 h. Quantitative data are shown as mean±SEM ( n = 3). One‐way ANOVA and Sidak's multiple comparisons test were performed. B) FACS analysis (left) and the quantification (right) of PD‐L1 levels on the surface of MC38 tumor cells treated with H 2 O 2 , Mn‐N/C, or plus with anti‐IFNAR1 antibody or NAC treatment for 24 h. Quantitative data are shown as mean±SEM ( n = 3). One‐way ANOVA and Sidak's multiple comparisons test were performed. C) Real‐time PCR analysis of PD‐L1 mRNA expression of tumor cells (RFP + ) sorted from MC38‐OVA tumor‐bearing mice with vehicle or Mn‐N/C treatment. Quantitative data are shown as mean±SEM ( n = 3). Two‐tailed unpaired t ‐test was performed for the comparisons between the two groups. D) FACS analysis (left) and the quantification (right) of PD‐L1 levels in CD45 − cells from MC38 tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 6 mice per group). Two‐tailed unpaired t ‐test was performed for the comparisons between the two groups. E) Schematic illustration of a bilateral model of MC38 tumors subcutaneously implanted on C57BL/6 mice and treated with vehicle or Mn‐N/C, or plus with anti‐PD‐L1 antibody. F,G) Tumor growth curve of the first tumor (F) and the second tumor (G) of MC38 in the WT mice treated as E described. Data are shown as mean±SEM ( n = 5 mice per group). Two‐way ANOVA (mixed model) and Sidak's multiple comparisons test were performed. H,I) FACS analysis (left) and the quantification (right) of tumor‐infiltrating CD8 + T cells in the first tumor (H) and the second tumor (I) of MC38 tumor‐bearing mice treated as E described. Data are shown as mean±SEM ( n = 5 mice per group). Two‐tailed unpaired t ‐test was performed for the comparisons between the two groups. J,K) FACS analysis (left) and the quantification (right) of Granzyme B + , PRF1 + , TNFα + of CD8 + T cells in the first tumor (J) and the second tumor (K) of MC38 tumor‐bearing mice treated as E described. Data are shown as mean±SEM ( n = 5 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; ns, no significance

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Two Tailed Test



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    Leinco Technologies gir-208 (neutralizing anti-human ifnar1)
    a Western blots for STING and VINCULIN in CRISPR/Cas9 control and STING knockout BP tumor cells (BP-sgControl and BP-sgSTING). Representative blots of two independent experiments are shown. b CellTiter-Glo analysis showing cell viability of BP-sgControl and BP-sgSTING cells after 3 days of treatment with serial dilution of olaparib ( n = 3). c Flow cytometry analysis of mouse BMDMs treated with DMSO vehicle control, olaparib (OL, 5 μM), 50% BP-sgSTING-CM, or 50% BP-sgSTING/OL-CM for two days ( n = 3). d ELISA analysis of IFNβ in media from BP-sgControl or BP-sgSTING cells with or without 2 days of olaparib treatment ( n = 7). e and f RT-qPCR analysis of Ccl5 ( e ) and Cxcl10 ( f ) in BP-sgControl and BP-sgSTING cells treated with or without olaparib for 2 days (BP-sgControl, n = 4; BP-sgSTING, n = 3). g Tumor growth (left) and survival (right) of BP-sgControl tumor-bearing FVB mice treated with olaparib (50 mg/kg, IP, QD), DMXAA (10 mg/kg, IP) or olaparib + DMXAA. Median survivals are shown in parentheses. Left, Control, n = 13; Olaparib, n = 7; DMXAA, n = 9; Olaparib + DMXAA, n = 14. Right, Control, n = 8; olaparib, n = 5; DMXAA, n = 6; olaparib + DMXAA, n = 9. h Tumor growth (left) and survival (right) of BP-sgSTING tumor-bearing FVB mice treated with olaparib (50 mg/kg, IP, QD), DMXAA (10 mg/kg, IP) or olaparib + DMXAA. Median survivals are shown in parentheses. Left, Control, n = 24; olaparib, n = 11; DMXAA, n = 9; olaparib + DMXAA, n = 19. Right, Control, n = 13; olaparib, n = 7; DMXAA, n = 6; olaparib + DMXAA, n = 11. i and j BP-sgControl ( i ) and BP-sgSTING ( j ) tumor growth in FVB mice treated with olaparib + DMXAA with or without anti-CD8 or <t>anti-IFNAR1</t> neutralizing antibodies ( n = 6 per condition). Data are presented as mean ± SEM. One-way ANOVA ( c, e , and f ). Two-tailed Mann–Whitney test ( d ). Two-way ANOVA for tumor growth ( g )–( j ). Log-rank Mantel–Cox test for survival ( g and h ). ns, not significant. Source data are provided as a Source Data file.
    Gir 208 (Neutralizing Anti Human Ifnar1), supplied by Leinco Technologies, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gir-208 (neutralizing anti-human ifnar1)/product/Leinco Technologies
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    monoclonal antibodies (mabs) gir-208 (neutralizing anti-human ifnar1)  (Leinco Technologies)
    90
    Leinco Technologies monoclonal antibodies (mabs) gir-208 (neutralizing anti-human ifnar1)
    a Western blots for STING and VINCULIN in CRISPR/Cas9 control and STING knockout BP tumor cells (BP-sgControl and BP-sgSTING). Representative blots of two independent experiments are shown. b CellTiter-Glo analysis showing cell viability of BP-sgControl and BP-sgSTING cells after 3 days of treatment with serial dilution of olaparib ( n = 3). c Flow cytometry analysis of mouse BMDMs treated with DMSO vehicle control, olaparib (OL, 5 μM), 50% BP-sgSTING-CM, or 50% BP-sgSTING/OL-CM for two days ( n = 3). d ELISA analysis of IFNβ in media from BP-sgControl or BP-sgSTING cells with or without 2 days of olaparib treatment ( n = 7). e and f RT-qPCR analysis of Ccl5 ( e ) and Cxcl10 ( f ) in BP-sgControl and BP-sgSTING cells treated with or without olaparib for 2 days (BP-sgControl, n = 4; BP-sgSTING, n = 3). g Tumor growth (left) and survival (right) of BP-sgControl tumor-bearing FVB mice treated with olaparib (50 mg/kg, IP, QD), DMXAA (10 mg/kg, IP) or olaparib + DMXAA. Median survivals are shown in parentheses. Left, Control, n = 13; Olaparib, n = 7; DMXAA, n = 9; Olaparib + DMXAA, n = 14. Right, Control, n = 8; olaparib, n = 5; DMXAA, n = 6; olaparib + DMXAA, n = 9. h Tumor growth (left) and survival (right) of BP-sgSTING tumor-bearing FVB mice treated with olaparib (50 mg/kg, IP, QD), DMXAA (10 mg/kg, IP) or olaparib + DMXAA. Median survivals are shown in parentheses. Left, Control, n = 24; olaparib, n = 11; DMXAA, n = 9; olaparib + DMXAA, n = 19. Right, Control, n = 13; olaparib, n = 7; DMXAA, n = 6; olaparib + DMXAA, n = 11. i and j BP-sgControl ( i ) and BP-sgSTING ( j ) tumor growth in FVB mice treated with olaparib + DMXAA with or without anti-CD8 or <t>anti-IFNAR1</t> neutralizing antibodies ( n = 6 per condition). Data are presented as mean ± SEM. One-way ANOVA ( c, e , and f ). Two-tailed Mann–Whitney test ( d ). Two-way ANOVA for tumor growth ( g )–( j ). Log-rank Mantel–Cox test for survival ( g and h ). ns, not significant. Source data are provided as a Source Data file.
    Monoclonal Antibodies (Mabs) Gir 208 (Neutralizing Anti Human Ifnar1), supplied by Leinco Technologies, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal antibodies (mabs) gir-208 (neutralizing anti-human ifnar1)/product/Leinco Technologies
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    anti-mouse ifnar1 neutralizing mab clone mar1-5a3  (Bio X Cell)
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    Bio X Cell anti-mouse ifnar1 neutralizing mab clone mar1-5a3
    IFN-β and KC are dispensable for IFI204-mediated host defense. IFI204 −/− mice were i.p. injected recombinant KC or IFN-β ( n = 10 each group) at a dose of 1.0 μg per mouse in 100 μL PBS on Day −1 and Day 0. The mice were infected intranasally with 1 × 10 8 CFU of Staphylococcus on Day 0. (A,B) Homogenized lung tissues were subjected to plating serial dilution for bacterial loads at 6 hpi or 24 hpi. (C) Blood were subjected to plating serial dilution for bacterial loads at 24 hpi. All data are shown as mean ± SEM. Student's t -test was performed. (D) The mice were i.p. injected with 2.5 mg anti-mouse <t>IFNAR1</t> neutralizing mAb or 2.5 mg IgG isotype control ( n = 8 each group), 24 h later, mice were i.v. challenged with 2 × 10 8 CFU of Staphylococcus . The animals were monitored daily for survival. *** p < 0.005.
    Anti Mouse Ifnar1 Neutralizing Mab Clone Mar1 5a3, supplied by Bio X Cell, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti-mouse ifnar1 neutralizing mab clone mar1-5a3/product/Bio X Cell
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    anti-ifnar1 neutralizing antibody  (Santa Cruz Biotechnology)
    90
    Santa Cruz Biotechnology anti-ifnar1 neutralizing antibody
    (A) Construct used to generate <t>cd2–ifnar1</t> transgenic mice. (B) RT-PCR demonstrated lymphoid tissue-specific expression of transgenic ifnar1. With the use of a reverse primer for FLAG, this PCR reaction amplified the ifnar1 transcript only from transgenic mice. Br, Brain; Liv, liver; Lu, lung; Sp, spleen; Th, thymus. (C) RNA was extracted from CD3+-enriched T cells, and ifnar1 mRNA was amplified by real-time RT-PCR. (D) Splenocytes from WT, Ifnar1−/−, and IFNAR1Texcl mice were stained for CD3, CD19, and IFNAR1. (E and F) Splenocytes were stimulated by IFN-α/β (250 U/ml) for 30 min (E) or for the indicated time (F) and then analyzed by Western blot. STAT1 and p-STAT1, 89/91 kDa; ISG15, 15 kDa; actin, 42 kDa. The images were spliced and joined, as indicated by white lines for the sake of the presentation. (G) Thymocytes were incubated with anti-IFNAR1-neutralizing antibody before IFN-α/β treatment and stained with a p-STAT1 antibody. The histogram plots show the p-STAT1-positive cells in the total live-cell gate (IFN treatment, bold, black line; antibody + IFN treatment, black line; no treatment, dotted line).
    Anti Ifnar1 Neutralizing Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti-ifnar1 neutralizing antibody/product/Santa Cruz Biotechnology
    Average 90 stars, based on 1 article reviews
    anti-ifnar1 neutralizing antibody - by Bioz Stars, 2026-03
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    anti-ifnar1 neutralizing antibody mar1-5a3  (Santa Cruz Biotechnology)
    90
    Santa Cruz Biotechnology anti-ifnar1 neutralizing antibody mar1-5a3
    (A) Construct used to generate <t>cd2–ifnar1</t> transgenic mice. (B) RT-PCR demonstrated lymphoid tissue-specific expression of transgenic ifnar1. With the use of a reverse primer for FLAG, this PCR reaction amplified the ifnar1 transcript only from transgenic mice. Br, Brain; Liv, liver; Lu, lung; Sp, spleen; Th, thymus. (C) RNA was extracted from CD3+-enriched T cells, and ifnar1 mRNA was amplified by real-time RT-PCR. (D) Splenocytes from WT, Ifnar1−/−, and IFNAR1Texcl mice were stained for CD3, CD19, and IFNAR1. (E and F) Splenocytes were stimulated by IFN-α/β (250 U/ml) for 30 min (E) or for the indicated time (F) and then analyzed by Western blot. STAT1 and p-STAT1, 89/91 kDa; ISG15, 15 kDa; actin, 42 kDa. The images were spliced and joined, as indicated by white lines for the sake of the presentation. (G) Thymocytes were incubated with anti-IFNAR1-neutralizing antibody before IFN-α/β treatment and stained with a p-STAT1 antibody. The histogram plots show the p-STAT1-positive cells in the total live-cell gate (IFN treatment, bold, black line; antibody + IFN treatment, black line; no treatment, dotted line).
    Anti Ifnar1 Neutralizing Antibody Mar1 5a3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti-ifnar1 neutralizing antibody mar1-5a3/product/Santa Cruz Biotechnology
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    anti-ifnar1 neutralizing antibody mar1-5a3 - by Bioz Stars, 2026-03
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    Image Search Results


    ( A ) Schematic representation of transgenic TRPV1 cre -GOF cKI mouse design. ( B and C ) IFN-α ( B ) and IFN-β ( C ) levels in DRG cultures of GOF ( n = 4) and TRPV1 cre -GOF ( n = 6) mice stimulated with ADU-S100 (1 μg/mL). ( D and E ) Measurement of thermal sensitivity of naive TRPV1 cre -GOF ( n = 15–16) and GOF ( n = 7–12) mice using the hot plate ( D ) or Hargreaves test ( E ). ( F ) Measurement of thermal withdrawal latency in hind paws of CFA-treated GOF ( n = 9) and TRPV1 cre -GOF ( n = 8) mice. ( G ) Measurement of thermal withdrawal latency in hind paws of CFA-treated TRPV1 cre -GOF mice that received either IgG control ( n = 5) or IFNAR1 neutralizing antibody (MAR1) before and 3 days after CFA injection ( n = 6). ( H ) Newly born TRPV1 cre -GOF pups (P5) were given 10 μL of AAV-PHP.S-DIO-IFNAR1-shRNA or AAV-PHP.S-DIO-scrambled-shRNA intraperitoneally. ( I ) Measurement of thermal sensitivity of mice injected with IFNAR1-Scr ( n = 9) or IFNAR1-shRNA ( n = 16) AAV using the hot plate. ( J ) Measurement of thermal withdrawal latency of CFA-treated mice infected with IFNAR1-Scr ( n = 7) or IFNAR1-shRNA ( n = 9) AAV. ( K ) Adult TRPV1 cre -GOF mice received 10 μL of AAV-DIO-IFNAR1-shRNA or AAV-DIO-scrambled-shRNA intrathecally. ( L ) Measurement of thermal withdrawal latency in hind paws of CFA-treated mice injected with IFNAR1-Scr ( n = 7) or IFNAR1-shRNA ( n = 8) AAV. Statistical analysis was performed using 1-way ANOVA followed by Tukey’s post hoc test ( B and C ; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001); t test ( E and I ) or Mann-Whitney test ( D ; ** P < 0.01, **** P < 0.0001); and 2-way ANOVA followed by Tukey’s post hoc test ( F : ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. TRPV1 cre -GOF i.l.; G : * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. TRPV1 cre -GOF +IgG i.l.; J and L : * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. IFNAR1-Scr i.l.; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001, $$$$ P < 0.0001 vs. IFNAR1-Scr c.l.).

    Journal: The Journal of Clinical Investigation

    Article Title: Induction of antiviral interferon-stimulated genes by neuronal STING promotes the resolution of pain in mice

    doi: 10.1172/JCI176474

    Figure Lengend Snippet: ( A ) Schematic representation of transgenic TRPV1 cre -GOF cKI mouse design. ( B and C ) IFN-α ( B ) and IFN-β ( C ) levels in DRG cultures of GOF ( n = 4) and TRPV1 cre -GOF ( n = 6) mice stimulated with ADU-S100 (1 μg/mL). ( D and E ) Measurement of thermal sensitivity of naive TRPV1 cre -GOF ( n = 15–16) and GOF ( n = 7–12) mice using the hot plate ( D ) or Hargreaves test ( E ). ( F ) Measurement of thermal withdrawal latency in hind paws of CFA-treated GOF ( n = 9) and TRPV1 cre -GOF ( n = 8) mice. ( G ) Measurement of thermal withdrawal latency in hind paws of CFA-treated TRPV1 cre -GOF mice that received either IgG control ( n = 5) or IFNAR1 neutralizing antibody (MAR1) before and 3 days after CFA injection ( n = 6). ( H ) Newly born TRPV1 cre -GOF pups (P5) were given 10 μL of AAV-PHP.S-DIO-IFNAR1-shRNA or AAV-PHP.S-DIO-scrambled-shRNA intraperitoneally. ( I ) Measurement of thermal sensitivity of mice injected with IFNAR1-Scr ( n = 9) or IFNAR1-shRNA ( n = 16) AAV using the hot plate. ( J ) Measurement of thermal withdrawal latency of CFA-treated mice infected with IFNAR1-Scr ( n = 7) or IFNAR1-shRNA ( n = 9) AAV. ( K ) Adult TRPV1 cre -GOF mice received 10 μL of AAV-DIO-IFNAR1-shRNA or AAV-DIO-scrambled-shRNA intrathecally. ( L ) Measurement of thermal withdrawal latency in hind paws of CFA-treated mice injected with IFNAR1-Scr ( n = 7) or IFNAR1-shRNA ( n = 8) AAV. Statistical analysis was performed using 1-way ANOVA followed by Tukey’s post hoc test ( B and C ; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001); t test ( E and I ) or Mann-Whitney test ( D ; ** P < 0.01, **** P < 0.0001); and 2-way ANOVA followed by Tukey’s post hoc test ( F : ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. TRPV1 cre -GOF i.l.; G : * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. TRPV1 cre -GOF +IgG i.l.; J and L : * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. IFNAR1-Scr i.l.; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001, $$$$ P < 0.0001 vs. IFNAR1-Scr c.l.).

    Article Snippet: Anti–mouse IFN-β neutralizing antibody (300 ng/mouse; PBL Assay Science, 32400-1), rabbit polyclonal IgG control (300 ng/mouse; BioLegend, CTL-4112), anti-mouse monoclonal IFNAR1 neutralizing antibody (MAR1; 1 μg/mouse; Leinco Technologies, I-401), and mouse IgG control (1 μg/mouse; Leinco Technologies, I-536) were dissolved in sterile PBS and administered intrathecally as described previously.

    Techniques: Transgenic Assay, Injection, shRNA, Infection, MANN-WHITNEY

    ( A ) Confocal image of TRPV1 neurons from TRPV1 Cre -GOF mice injected with DIO-Scr-shRNA ( n = 5) or DIO-IFNAR1-shRNA AAVs ( n = 5). Images represent DAPI staining, AAV-GFP expression (green), and Kchip1 transcripts (red) by RNAscope. Scale bars: 25 μm, and 10 μm on cropped images. ( B ) Quantification of Kchip1 density measured by the number of transcripts represented by dots per surface unit in AAV-infected TRPV1 neurons. ( C ) Representative current clamp recording of TdTomato + /GFP + TRPV1 neurons from TRPV1 Cre -GOF mice infected with DIO-Scr-shRNA or DIO-IFNAR1-shRNA AAVs (top). Cells were injected with 500-millisecond current pulses with an increment of 10 pA and an interval of 5 seconds (protocol, bottom). The highlighted black line indicates the current amplitude that induces the first AP. Scale bars: 20 mV/50 ms. ( D ) Measurement of rheobase in infected (GFP + ) and non-infected (GFP – ) TRPV1 neurons (TdTomato + ) recorded in C . Data are presented as dot plots with mean values ( IFNAR1-Scr neurons, n = 89; IFNAR1-shRNA –infected neurons, n = 80; IFNAR1-Scr neurons, n = 18; IFNAR1-shRNA –non-infected neurons, n = 18). ( E ) Number of spikes evoked by injected current in TRPV1 (TdTomato + ) and AAV-infected (GFP + ) neurons. ( F ) Representative capsaicin-evoked current (100 nM) in TdTomato + /GFP + TRPV1 neurons from TRPV1 Cre -GOF mice infected with IFNAR1-Scr and IFNAR1-shRNA AAVs. Scale bars: 200 pA/20 s. ( G ) Current density evoked by capsaicin in cells represented in F ( IFNAR1-Scr neurons, n = 41; IFNAR1-shRNA neurons, n = 28). ( H ) Representative outward potassium currents recorded in response to voltage steps in TRPV1 Cre -GOF neurons infected with IFNAR1-Scr and IFNAR1-shRNA AAVs. Scale bars: 1 nA/100 ms. ( I ) Average current-voltage relationship in TRPV1 neurons from TRPV1 Cre -GOF mice infected with IFNAR1-Scr ( n = 31) or IFNAR1-shRNA AAVs ( n = 25). Statistical analysis was performed using t test ( B ) or Mann-Whitney test ( D and G ; **** P < 0.0001) and 2-way ANOVA with Tukey’s post hoc test ( E and I ; ** P < 0.01, **** P < 0.0001).

    Journal: The Journal of Clinical Investigation

    Article Title: Induction of antiviral interferon-stimulated genes by neuronal STING promotes the resolution of pain in mice

    doi: 10.1172/JCI176474

    Figure Lengend Snippet: ( A ) Confocal image of TRPV1 neurons from TRPV1 Cre -GOF mice injected with DIO-Scr-shRNA ( n = 5) or DIO-IFNAR1-shRNA AAVs ( n = 5). Images represent DAPI staining, AAV-GFP expression (green), and Kchip1 transcripts (red) by RNAscope. Scale bars: 25 μm, and 10 μm on cropped images. ( B ) Quantification of Kchip1 density measured by the number of transcripts represented by dots per surface unit in AAV-infected TRPV1 neurons. ( C ) Representative current clamp recording of TdTomato + /GFP + TRPV1 neurons from TRPV1 Cre -GOF mice infected with DIO-Scr-shRNA or DIO-IFNAR1-shRNA AAVs (top). Cells were injected with 500-millisecond current pulses with an increment of 10 pA and an interval of 5 seconds (protocol, bottom). The highlighted black line indicates the current amplitude that induces the first AP. Scale bars: 20 mV/50 ms. ( D ) Measurement of rheobase in infected (GFP + ) and non-infected (GFP – ) TRPV1 neurons (TdTomato + ) recorded in C . Data are presented as dot plots with mean values ( IFNAR1-Scr neurons, n = 89; IFNAR1-shRNA –infected neurons, n = 80; IFNAR1-Scr neurons, n = 18; IFNAR1-shRNA –non-infected neurons, n = 18). ( E ) Number of spikes evoked by injected current in TRPV1 (TdTomato + ) and AAV-infected (GFP + ) neurons. ( F ) Representative capsaicin-evoked current (100 nM) in TdTomato + /GFP + TRPV1 neurons from TRPV1 Cre -GOF mice infected with IFNAR1-Scr and IFNAR1-shRNA AAVs. Scale bars: 200 pA/20 s. ( G ) Current density evoked by capsaicin in cells represented in F ( IFNAR1-Scr neurons, n = 41; IFNAR1-shRNA neurons, n = 28). ( H ) Representative outward potassium currents recorded in response to voltage steps in TRPV1 Cre -GOF neurons infected with IFNAR1-Scr and IFNAR1-shRNA AAVs. Scale bars: 1 nA/100 ms. ( I ) Average current-voltage relationship in TRPV1 neurons from TRPV1 Cre -GOF mice infected with IFNAR1-Scr ( n = 31) or IFNAR1-shRNA AAVs ( n = 25). Statistical analysis was performed using t test ( B ) or Mann-Whitney test ( D and G ; **** P < 0.0001) and 2-way ANOVA with Tukey’s post hoc test ( E and I ; ** P < 0.01, **** P < 0.0001).

    Article Snippet: Anti–mouse IFN-β neutralizing antibody (300 ng/mouse; PBL Assay Science, 32400-1), rabbit polyclonal IgG control (300 ng/mouse; BioLegend, CTL-4112), anti-mouse monoclonal IFNAR1 neutralizing antibody (MAR1; 1 μg/mouse; Leinco Technologies, I-401), and mouse IgG control (1 μg/mouse; Leinco Technologies, I-536) were dissolved in sterile PBS and administered intrathecally as described previously.

    Techniques: Injection, shRNA, Staining, Expressing, Infection, MANN-WHITNEY

    Type I interferon signaling is critical for Mn‐N/C‐mediated anti‐tumor immune response. A) FACS analysis (left) of CFSE and the quantification (right) of OT‐1 T cell proliferation after co‐cultured with DCs sorted from lymph nodes of WT and Ifnar1 −/− MC38‐OVA tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 4 per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. B) FACS analysis (below) and the quantification (above) of IFNγ + , Granzyme B + , TNFα + of OT‐1 CD8 + T cells after co‐culture with DCs as A described. Data are shown as mean±SEM ( n = 4 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. C) FACS analysis (above) and the quantification (below) of tumor‐infiltrating CD8 + T cells in WT and Ifnar1 −/− MC38 tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 5–6 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. D) FACS analysis (right) and the quantification (left) of IFNγ + , PRF1 + (Perforin), TNFα + of CD8 + T cells in WT and Ifnar1 −/− MC38 tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 5–6 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. E) Tumor growth curve of MC38 in WT and Ifnar1 −/− mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 5–6 mice per group). Two‐way ANOVA (mixed model) and Sidak's multiple comparisons test were performed. F) MC38 tumor weight in WT and Ifnar1 −/− mice with vehicle or Mn‐N/C treatment 14 days after tumor cell inoculation. Data are shown as mean±SEM ( n = 5–6 mice per group). G) Kaplan–Meier analysis of WT and Ifnar1 −/− mice survival with vehicle or Mn‐N/C treatment ( n = 5–6 mice per group). * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; ns, no significance

    Journal: Advanced Science

    Article Title: A Single‐Atom Manganese Nanozyme Mn‐N/C Promotes Anti‐Tumor Immune Response via Eliciting Type I Interferon Signaling

    doi: 10.1002/advs.202305979

    Figure Lengend Snippet: Type I interferon signaling is critical for Mn‐N/C‐mediated anti‐tumor immune response. A) FACS analysis (left) of CFSE and the quantification (right) of OT‐1 T cell proliferation after co‐cultured with DCs sorted from lymph nodes of WT and Ifnar1 −/− MC38‐OVA tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 4 per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. B) FACS analysis (below) and the quantification (above) of IFNγ + , Granzyme B + , TNFα + of OT‐1 CD8 + T cells after co‐culture with DCs as A described. Data are shown as mean±SEM ( n = 4 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. C) FACS analysis (above) and the quantification (below) of tumor‐infiltrating CD8 + T cells in WT and Ifnar1 −/− MC38 tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 5–6 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. D) FACS analysis (right) and the quantification (left) of IFNγ + , PRF1 + (Perforin), TNFα + of CD8 + T cells in WT and Ifnar1 −/− MC38 tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 5–6 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. E) Tumor growth curve of MC38 in WT and Ifnar1 −/− mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 5–6 mice per group). Two‐way ANOVA (mixed model) and Sidak's multiple comparisons test were performed. F) MC38 tumor weight in WT and Ifnar1 −/− mice with vehicle or Mn‐N/C treatment 14 days after tumor cell inoculation. Data are shown as mean±SEM ( n = 5–6 mice per group). G) Kaplan–Meier analysis of WT and Ifnar1 −/− mice survival with vehicle or Mn‐N/C treatment ( n = 5–6 mice per group). * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; ns, no significance

    Article Snippet: Other inhibitors including 1 µ m STING inhibitor H151 (TargetMol, cat#T5674), or 20 µg mL −1 anti‐IFNAR1 neutralizing antibody (BioXCell, cat#BE0241) were added together with Mn‐N/C and H 2 O 2 .

    Techniques: Cell Culture, Co-Culture Assay

    A combination of Mn‐N/C and PD‐L1 blockade synergistically suppresses tumor growth. A) Real‐time PCR analysis of PD‐L1 mRNA expression of MC38 tumor cells treated with H 2 O 2 , Mn‐N/C for 24 h. Quantitative data are shown as mean±SEM ( n = 3). One‐way ANOVA and Sidak's multiple comparisons test were performed. B) FACS analysis (left) and the quantification (right) of PD‐L1 levels on the surface of MC38 tumor cells treated with H 2 O 2 , Mn‐N/C, or plus with anti‐IFNAR1 antibody or NAC treatment for 24 h. Quantitative data are shown as mean±SEM ( n = 3). One‐way ANOVA and Sidak's multiple comparisons test were performed. C) Real‐time PCR analysis of PD‐L1 mRNA expression of tumor cells (RFP + ) sorted from MC38‐OVA tumor‐bearing mice with vehicle or Mn‐N/C treatment. Quantitative data are shown as mean±SEM ( n = 3). Two‐tailed unpaired t ‐test was performed for the comparisons between the two groups. D) FACS analysis (left) and the quantification (right) of PD‐L1 levels in CD45 − cells from MC38 tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 6 mice per group). Two‐tailed unpaired t ‐test was performed for the comparisons between the two groups. E) Schematic illustration of a bilateral model of MC38 tumors subcutaneously implanted on C57BL/6 mice and treated with vehicle or Mn‐N/C, or plus with anti‐PD‐L1 antibody. F,G) Tumor growth curve of the first tumor (F) and the second tumor (G) of MC38 in the WT mice treated as E described. Data are shown as mean±SEM ( n = 5 mice per group). Two‐way ANOVA (mixed model) and Sidak's multiple comparisons test were performed. H,I) FACS analysis (left) and the quantification (right) of tumor‐infiltrating CD8 + T cells in the first tumor (H) and the second tumor (I) of MC38 tumor‐bearing mice treated as E described. Data are shown as mean±SEM ( n = 5 mice per group). Two‐tailed unpaired t ‐test was performed for the comparisons between the two groups. J,K) FACS analysis (left) and the quantification (right) of Granzyme B + , PRF1 + , TNFα + of CD8 + T cells in the first tumor (J) and the second tumor (K) of MC38 tumor‐bearing mice treated as E described. Data are shown as mean±SEM ( n = 5 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; ns, no significance

    Journal: Advanced Science

    Article Title: A Single‐Atom Manganese Nanozyme Mn‐N/C Promotes Anti‐Tumor Immune Response via Eliciting Type I Interferon Signaling

    doi: 10.1002/advs.202305979

    Figure Lengend Snippet: A combination of Mn‐N/C and PD‐L1 blockade synergistically suppresses tumor growth. A) Real‐time PCR analysis of PD‐L1 mRNA expression of MC38 tumor cells treated with H 2 O 2 , Mn‐N/C for 24 h. Quantitative data are shown as mean±SEM ( n = 3). One‐way ANOVA and Sidak's multiple comparisons test were performed. B) FACS analysis (left) and the quantification (right) of PD‐L1 levels on the surface of MC38 tumor cells treated with H 2 O 2 , Mn‐N/C, or plus with anti‐IFNAR1 antibody or NAC treatment for 24 h. Quantitative data are shown as mean±SEM ( n = 3). One‐way ANOVA and Sidak's multiple comparisons test were performed. C) Real‐time PCR analysis of PD‐L1 mRNA expression of tumor cells (RFP + ) sorted from MC38‐OVA tumor‐bearing mice with vehicle or Mn‐N/C treatment. Quantitative data are shown as mean±SEM ( n = 3). Two‐tailed unpaired t ‐test was performed for the comparisons between the two groups. D) FACS analysis (left) and the quantification (right) of PD‐L1 levels in CD45 − cells from MC38 tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 6 mice per group). Two‐tailed unpaired t ‐test was performed for the comparisons between the two groups. E) Schematic illustration of a bilateral model of MC38 tumors subcutaneously implanted on C57BL/6 mice and treated with vehicle or Mn‐N/C, or plus with anti‐PD‐L1 antibody. F,G) Tumor growth curve of the first tumor (F) and the second tumor (G) of MC38 in the WT mice treated as E described. Data are shown as mean±SEM ( n = 5 mice per group). Two‐way ANOVA (mixed model) and Sidak's multiple comparisons test were performed. H,I) FACS analysis (left) and the quantification (right) of tumor‐infiltrating CD8 + T cells in the first tumor (H) and the second tumor (I) of MC38 tumor‐bearing mice treated as E described. Data are shown as mean±SEM ( n = 5 mice per group). Two‐tailed unpaired t ‐test was performed for the comparisons between the two groups. J,K) FACS analysis (left) and the quantification (right) of Granzyme B + , PRF1 + , TNFα + of CD8 + T cells in the first tumor (J) and the second tumor (K) of MC38 tumor‐bearing mice treated as E described. Data are shown as mean±SEM ( n = 5 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; ns, no significance

    Article Snippet: Other inhibitors including 1 µ m STING inhibitor H151 (TargetMol, cat#T5674), or 20 µg mL −1 anti‐IFNAR1 neutralizing antibody (BioXCell, cat#BE0241) were added together with Mn‐N/C and H 2 O 2 .

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Two Tailed Test

    a Western blots for STING and VINCULIN in CRISPR/Cas9 control and STING knockout BP tumor cells (BP-sgControl and BP-sgSTING). Representative blots of two independent experiments are shown. b CellTiter-Glo analysis showing cell viability of BP-sgControl and BP-sgSTING cells after 3 days of treatment with serial dilution of olaparib ( n = 3). c Flow cytometry analysis of mouse BMDMs treated with DMSO vehicle control, olaparib (OL, 5 μM), 50% BP-sgSTING-CM, or 50% BP-sgSTING/OL-CM for two days ( n = 3). d ELISA analysis of IFNβ in media from BP-sgControl or BP-sgSTING cells with or without 2 days of olaparib treatment ( n = 7). e and f RT-qPCR analysis of Ccl5 ( e ) and Cxcl10 ( f ) in BP-sgControl and BP-sgSTING cells treated with or without olaparib for 2 days (BP-sgControl, n = 4; BP-sgSTING, n = 3). g Tumor growth (left) and survival (right) of BP-sgControl tumor-bearing FVB mice treated with olaparib (50 mg/kg, IP, QD), DMXAA (10 mg/kg, IP) or olaparib + DMXAA. Median survivals are shown in parentheses. Left, Control, n = 13; Olaparib, n = 7; DMXAA, n = 9; Olaparib + DMXAA, n = 14. Right, Control, n = 8; olaparib, n = 5; DMXAA, n = 6; olaparib + DMXAA, n = 9. h Tumor growth (left) and survival (right) of BP-sgSTING tumor-bearing FVB mice treated with olaparib (50 mg/kg, IP, QD), DMXAA (10 mg/kg, IP) or olaparib + DMXAA. Median survivals are shown in parentheses. Left, Control, n = 24; olaparib, n = 11; DMXAA, n = 9; olaparib + DMXAA, n = 19. Right, Control, n = 13; olaparib, n = 7; DMXAA, n = 6; olaparib + DMXAA, n = 11. i and j BP-sgControl ( i ) and BP-sgSTING ( j ) tumor growth in FVB mice treated with olaparib + DMXAA with or without anti-CD8 or anti-IFNAR1 neutralizing antibodies ( n = 6 per condition). Data are presented as mean ± SEM. One-way ANOVA ( c, e , and f ). Two-tailed Mann–Whitney test ( d ). Two-way ANOVA for tumor growth ( g )–( j ). Log-rank Mantel–Cox test for survival ( g and h ). ns, not significant. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: STING agonism reprograms tumor-associated macrophages and overcomes resistance to PARP inhibition in BRCA1-deficient models of breast cancer

    doi: 10.1038/s41467-022-30568-1

    Figure Lengend Snippet: a Western blots for STING and VINCULIN in CRISPR/Cas9 control and STING knockout BP tumor cells (BP-sgControl and BP-sgSTING). Representative blots of two independent experiments are shown. b CellTiter-Glo analysis showing cell viability of BP-sgControl and BP-sgSTING cells after 3 days of treatment with serial dilution of olaparib ( n = 3). c Flow cytometry analysis of mouse BMDMs treated with DMSO vehicle control, olaparib (OL, 5 μM), 50% BP-sgSTING-CM, or 50% BP-sgSTING/OL-CM for two days ( n = 3). d ELISA analysis of IFNβ in media from BP-sgControl or BP-sgSTING cells with or without 2 days of olaparib treatment ( n = 7). e and f RT-qPCR analysis of Ccl5 ( e ) and Cxcl10 ( f ) in BP-sgControl and BP-sgSTING cells treated with or without olaparib for 2 days (BP-sgControl, n = 4; BP-sgSTING, n = 3). g Tumor growth (left) and survival (right) of BP-sgControl tumor-bearing FVB mice treated with olaparib (50 mg/kg, IP, QD), DMXAA (10 mg/kg, IP) or olaparib + DMXAA. Median survivals are shown in parentheses. Left, Control, n = 13; Olaparib, n = 7; DMXAA, n = 9; Olaparib + DMXAA, n = 14. Right, Control, n = 8; olaparib, n = 5; DMXAA, n = 6; olaparib + DMXAA, n = 9. h Tumor growth (left) and survival (right) of BP-sgSTING tumor-bearing FVB mice treated with olaparib (50 mg/kg, IP, QD), DMXAA (10 mg/kg, IP) or olaparib + DMXAA. Median survivals are shown in parentheses. Left, Control, n = 24; olaparib, n = 11; DMXAA, n = 9; olaparib + DMXAA, n = 19. Right, Control, n = 13; olaparib, n = 7; DMXAA, n = 6; olaparib + DMXAA, n = 11. i and j BP-sgControl ( i ) and BP-sgSTING ( j ) tumor growth in FVB mice treated with olaparib + DMXAA with or without anti-CD8 or anti-IFNAR1 neutralizing antibodies ( n = 6 per condition). Data are presented as mean ± SEM. One-way ANOVA ( c, e , and f ). Two-tailed Mann–Whitney test ( d ). Two-way ANOVA for tumor growth ( g )–( j ). Log-rank Mantel–Cox test for survival ( g and h ). ns, not significant. Source data are provided as a Source Data file.

    Article Snippet: For IFNAR1 blockade, anti-mouse IFNAR1 neutralizing antibody (200 μg/mouse; clone MAR1-5A3; BioXcell, # BE0241) was administered via IP 72 h and 24 h before start of the combination therapy (olaparib + DMXAA) and every 3 days thereafter.

    Techniques: Western Blot, CRISPR, Control, Knock-Out, Serial Dilution, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY

    IFN-β and KC are dispensable for IFI204-mediated host defense. IFI204 −/− mice were i.p. injected recombinant KC or IFN-β ( n = 10 each group) at a dose of 1.0 μg per mouse in 100 μL PBS on Day −1 and Day 0. The mice were infected intranasally with 1 × 10 8 CFU of Staphylococcus on Day 0. (A,B) Homogenized lung tissues were subjected to plating serial dilution for bacterial loads at 6 hpi or 24 hpi. (C) Blood were subjected to plating serial dilution for bacterial loads at 24 hpi. All data are shown as mean ± SEM. Student's t -test was performed. (D) The mice were i.p. injected with 2.5 mg anti-mouse IFNAR1 neutralizing mAb or 2.5 mg IgG isotype control ( n = 8 each group), 24 h later, mice were i.v. challenged with 2 × 10 8 CFU of Staphylococcus . The animals were monitored daily for survival. *** p < 0.005.

    Journal: Frontiers in Immunology

    Article Title: DNA Sensor IFI204 Contributes to Host Defense Against Staphylococcus aureus Infection in Mice

    doi: 10.3389/fimmu.2019.00474

    Figure Lengend Snippet: IFN-β and KC are dispensable for IFI204-mediated host defense. IFI204 −/− mice were i.p. injected recombinant KC or IFN-β ( n = 10 each group) at a dose of 1.0 μg per mouse in 100 μL PBS on Day −1 and Day 0. The mice were infected intranasally with 1 × 10 8 CFU of Staphylococcus on Day 0. (A,B) Homogenized lung tissues were subjected to plating serial dilution for bacterial loads at 6 hpi or 24 hpi. (C) Blood were subjected to plating serial dilution for bacterial loads at 24 hpi. All data are shown as mean ± SEM. Student's t -test was performed. (D) The mice were i.p. injected with 2.5 mg anti-mouse IFNAR1 neutralizing mAb or 2.5 mg IgG isotype control ( n = 8 each group), 24 h later, mice were i.v. challenged with 2 × 10 8 CFU of Staphylococcus . The animals were monitored daily for survival. *** p < 0.005.

    Article Snippet: Mice were i.p. inoculated with 2.5 mg anti-mouse IFNAR1 neutralizing mAb (clone MAR1-5A3, BioXcell) or 2.5 mg IgG isotype control (Clone MOPC-21, BioXcell).

    Techniques: Injection, Recombinant, Infection, Serial Dilution, Control

    (A) Construct used to generate cd2–ifnar1 transgenic mice. (B) RT-PCR demonstrated lymphoid tissue-specific expression of transgenic ifnar1. With the use of a reverse primer for FLAG, this PCR reaction amplified the ifnar1 transcript only from transgenic mice. Br, Brain; Liv, liver; Lu, lung; Sp, spleen; Th, thymus. (C) RNA was extracted from CD3+-enriched T cells, and ifnar1 mRNA was amplified by real-time RT-PCR. (D) Splenocytes from WT, Ifnar1−/−, and IFNAR1Texcl mice were stained for CD3, CD19, and IFNAR1. (E and F) Splenocytes were stimulated by IFN-α/β (250 U/ml) for 30 min (E) or for the indicated time (F) and then analyzed by Western blot. STAT1 and p-STAT1, 89/91 kDa; ISG15, 15 kDa; actin, 42 kDa. The images were spliced and joined, as indicated by white lines for the sake of the presentation. (G) Thymocytes were incubated with anti-IFNAR1-neutralizing antibody before IFN-α/β treatment and stained with a p-STAT1 antibody. The histogram plots show the p-STAT1-positive cells in the total live-cell gate (IFN treatment, bold, black line; antibody + IFN treatment, black line; no treatment, dotted line).

    Journal: Journal of Leukocyte Biology

    Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

    doi: 10.1189/jlb.3A1214-598R

    Figure Lengend Snippet: (A) Construct used to generate cd2–ifnar1 transgenic mice. (B) RT-PCR demonstrated lymphoid tissue-specific expression of transgenic ifnar1. With the use of a reverse primer for FLAG, this PCR reaction amplified the ifnar1 transcript only from transgenic mice. Br, Brain; Liv, liver; Lu, lung; Sp, spleen; Th, thymus. (C) RNA was extracted from CD3+-enriched T cells, and ifnar1 mRNA was amplified by real-time RT-PCR. (D) Splenocytes from WT, Ifnar1−/−, and IFNAR1Texcl mice were stained for CD3, CD19, and IFNAR1. (E and F) Splenocytes were stimulated by IFN-α/β (250 U/ml) for 30 min (E) or for the indicated time (F) and then analyzed by Western blot. STAT1 and p-STAT1, 89/91 kDa; ISG15, 15 kDa; actin, 42 kDa. The images were spliced and joined, as indicated by white lines for the sake of the presentation. (G) Thymocytes were incubated with anti-IFNAR1-neutralizing antibody before IFN-α/β treatment and stained with a p-STAT1 antibody. The histogram plots show the p-STAT1-positive cells in the total live-cell gate (IFN treatment, bold, black line; antibody + IFN treatment, black line; no treatment, dotted line).

    Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

    Techniques: Construct, Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Quantitative RT-PCR, Staining, Western Blot, Incubation

    (A–C) Splenocytes (A), peritoneal macrophages (B), and LN cells (C) were isolated from WT, Ifnar1−/−, and IFNAR1Texcl mice. Cells were treated with IFN-α/β (250 U/ml) for 15 min and double stained for CD3, CD4, CD8, CD11b, CD19, and p-STAT1. Dot plots show the double-positive cells in the total live-cell gate, and a representative dot plot/group is shown. (D and E) Thymidine uptake after anti (a)-CD3/anti-CD28 stimulation of splenocytes (D) and LN cells (E). Results shown are presented as mean values ± sem. (F) Cytokine production in splenocyte supernatants following stimulation with anti-CD3/anti-CD28. (G) Intracellular staining of splenocytes for IL-2 and IFN-γ after stimulation with anti-CD3/anti-CD28, followed by PMA/ionomycin/brefeldin A incubation. (H and I) CD3+-enriched T cells were stimulated under Th1- and Th17-polarizing conditions and stained for IFN-γ (H) and IL-17A (I). The bars represent the double-positive (CD4+ cytokine+) cells in the total live-cell gate. All data shown are representative of at least 2 independent experiments (n = 5 mice/genotype/experiment).

    Journal: Journal of Leukocyte Biology

    Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

    doi: 10.1189/jlb.3A1214-598R

    Figure Lengend Snippet: (A–C) Splenocytes (A), peritoneal macrophages (B), and LN cells (C) were isolated from WT, Ifnar1−/−, and IFNAR1Texcl mice. Cells were treated with IFN-α/β (250 U/ml) for 15 min and double stained for CD3, CD4, CD8, CD11b, CD19, and p-STAT1. Dot plots show the double-positive cells in the total live-cell gate, and a representative dot plot/group is shown. (D and E) Thymidine uptake after anti (a)-CD3/anti-CD28 stimulation of splenocytes (D) and LN cells (E). Results shown are presented as mean values ± sem. (F) Cytokine production in splenocyte supernatants following stimulation with anti-CD3/anti-CD28. (G) Intracellular staining of splenocytes for IL-2 and IFN-γ after stimulation with anti-CD3/anti-CD28, followed by PMA/ionomycin/brefeldin A incubation. (H and I) CD3+-enriched T cells were stimulated under Th1- and Th17-polarizing conditions and stained for IFN-γ (H) and IL-17A (I). The bars represent the double-positive (CD4+ cytokine+) cells in the total live-cell gate. All data shown are representative of at least 2 independent experiments (n = 5 mice/genotype/experiment).

    Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

    Techniques: Isolation, Staining, Incubation

    (A) Mean clinical scores for WT, Ifnar1−/−, and IFNAR1Texcl mice (n = 9 mice/genotype) after immunization with MOG35–55 peptide. Results shown are representative of 3 independent experiments and are presented as mean values ± sem. (B) Mean body weight of WT (n = 30), Ifnar1−/− (n = 28), and IFNAR1Texcl (n = 32) mice after EAE induction, pooled from 3 independent experiments. Results shown are presented as mean values ± sem.*P < 0.05, **P < 0.01: IFNAR1Texcl versus WT mice; and +P < 0.05, ++P < 0.01: IFNAR1Texcl versus Ifnar1−/− mice.

    Journal: Journal of Leukocyte Biology

    Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

    doi: 10.1189/jlb.3A1214-598R

    Figure Lengend Snippet: (A) Mean clinical scores for WT, Ifnar1−/−, and IFNAR1Texcl mice (n = 9 mice/genotype) after immunization with MOG35–55 peptide. Results shown are representative of 3 independent experiments and are presented as mean values ± sem. (B) Mean body weight of WT (n = 30), Ifnar1−/− (n = 28), and IFNAR1Texcl (n = 32) mice after EAE induction, pooled from 3 independent experiments. Results shown are presented as mean values ± sem.*P < 0.05, **P < 0.01: IFNAR1Texcl versus WT mice; and +P < 0.05, ++P < 0.01: IFNAR1Texcl versus Ifnar1−/− mice.

    Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

    Techniques:

    Clinical severity, day of onset, and peak of MOG 35–55 -induced EAE in IFNAR1 Texcl and control mice

    Journal: Journal of Leukocyte Biology

    Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

    doi: 10.1189/jlb.3A1214-598R

    Figure Lengend Snippet: Clinical severity, day of onset, and peak of MOG 35–55 -induced EAE in IFNAR1 Texcl and control mice

    Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

    Techniques:

    Clinical severity of WT and IFNAR1 Texcl mice upon PBS or IFN-β administration

    Journal: Journal of Leukocyte Biology

    Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

    doi: 10.1189/jlb.3A1214-598R

    Figure Lengend Snippet: Clinical severity of WT and IFNAR1 Texcl mice upon PBS or IFN-β administration

    Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

    Techniques:

    (A–D) Spinal cord sections from WT, Ifnar1−/−, and IFNAR1Texcl mice (n = 3 mice/genotype) were prepared 17 d upon EAE induction and stained with H&E (A), Luxol fast blue (B), and Bielschowsky's silver stain (C). Immunohistochemistry for CD3+ T cells was also performed (D). Arrows indicate immune cell infiltration (A), demyelination (B), axonal damage (C), and CD3+ T cell infiltration in the CNS parenchyma. Scale bars, 1 mm (A–C); 200 μm (D).

    Journal: Journal of Leukocyte Biology

    Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

    doi: 10.1189/jlb.3A1214-598R

    Figure Lengend Snippet: (A–D) Spinal cord sections from WT, Ifnar1−/−, and IFNAR1Texcl mice (n = 3 mice/genotype) were prepared 17 d upon EAE induction and stained with H&E (A), Luxol fast blue (B), and Bielschowsky's silver stain (C). Immunohistochemistry for CD3+ T cells was also performed (D). Arrows indicate immune cell infiltration (A), demyelination (B), axonal damage (C), and CD3+ T cell infiltration in the CNS parenchyma. Scale bars, 1 mm (A–C); 200 μm (D).

    Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

    Techniques: Staining, Silver Staining, Immunohistochemistry

    Splenocytes were isolated from WT, Ifnar1−/−, and IFNAR1Texcl mice, 10 d upon EAE induction. (A) Cells were stimulated with PMA/ionomycin/brefeldin A, and the frequency of IL- 17A-producing CD4+ T cells in the spleen was determined. The dot plots and bars represent IL-17A+ cells gated on total splenocytes. A representative dot plot per genotype is shown. (B) Expression levels of IL-17A by CD4+ T cells were evaluated by measuring MFI from (A) dot plots. (C) Splenocytes from all groups were restimulated ex vivo with MOG35–55 for 72 h, and the levels of secreted IL-17A were measured. (D) Frequency of CCR6+ splenocytes was determined (gated on the CD4+ compartment). All results are shown as means ± sem (n = 5 mice/genotype) and are representative from 2 independent experiments with similar results. *P < 0.05, **P < 0.01, ***P < 0.001.

    Journal: Journal of Leukocyte Biology

    Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

    doi: 10.1189/jlb.3A1214-598R

    Figure Lengend Snippet: Splenocytes were isolated from WT, Ifnar1−/−, and IFNAR1Texcl mice, 10 d upon EAE induction. (A) Cells were stimulated with PMA/ionomycin/brefeldin A, and the frequency of IL- 17A-producing CD4+ T cells in the spleen was determined. The dot plots and bars represent IL-17A+ cells gated on total splenocytes. A representative dot plot per genotype is shown. (B) Expression levels of IL-17A by CD4+ T cells were evaluated by measuring MFI from (A) dot plots. (C) Splenocytes from all groups were restimulated ex vivo with MOG35–55 for 72 h, and the levels of secreted IL-17A were measured. (D) Frequency of CCR6+ splenocytes was determined (gated on the CD4+ compartment). All results are shown as means ± sem (n = 5 mice/genotype) and are representative from 2 independent experiments with similar results. *P < 0.05, **P < 0.01, ***P < 0.001.

    Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

    Techniques: Isolation, Expressing, Ex Vivo

    Microarray analysis was performed on RNA from splenic, CD3+-enriched T cells and spinal cords from WT, Ifnar1−/−, and IFNAR1Texcl mice on d 0, 10, and 17 relative to EAE induction. (A and B) Heat map depicting the relative expression of selected genes that are different in expression between IFNAR1Texcl and WT T cells (A) and spinal cord (B) samples and were common in 2 independent experiments. Table 4 shows the function and number of molecules/category, as assessed by DAVID microarray software. The levels of mRNA transcripts for selected genes were measured in T cells (C) and spinal cords (D–F) by quantitative RT-PCR by use of single samples from each group at each time point (d 0: n = 3–5 mice/genotype; d 10: n = 3–4 mice/genotype; d 17: n = 2–3 mice/genotype). The results shown are the means ± sem of samples from 1 representative of 2 independent EAE experiments with similar results.*P < 0.05, **P < 0.01, ***P < 0.001.

    Journal: Journal of Leukocyte Biology

    Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

    doi: 10.1189/jlb.3A1214-598R

    Figure Lengend Snippet: Microarray analysis was performed on RNA from splenic, CD3+-enriched T cells and spinal cords from WT, Ifnar1−/−, and IFNAR1Texcl mice on d 0, 10, and 17 relative to EAE induction. (A and B) Heat map depicting the relative expression of selected genes that are different in expression between IFNAR1Texcl and WT T cells (A) and spinal cord (B) samples and were common in 2 independent experiments. Table 4 shows the function and number of molecules/category, as assessed by DAVID microarray software. The levels of mRNA transcripts for selected genes were measured in T cells (C) and spinal cords (D–F) by quantitative RT-PCR by use of single samples from each group at each time point (d 0: n = 3–5 mice/genotype; d 10: n = 3–4 mice/genotype; d 17: n = 2–3 mice/genotype). The results shown are the means ± sem of samples from 1 representative of 2 independent EAE experiments with similar results.*P < 0.05, **P < 0.01, ***P < 0.001.

    Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

    Techniques: Microarray, Expressing, Software, Quantitative RT-PCR

    (A) Construct used to generate cd2–ifnar1 transgenic mice. (B) RT-PCR demonstrated lymphoid tissue-specific expression of transgenic ifnar1. With the use of a reverse primer for FLAG, this PCR reaction amplified the ifnar1 transcript only from transgenic mice. Br, Brain; Liv, liver; Lu, lung; Sp, spleen; Th, thymus. (C) RNA was extracted from CD3+-enriched T cells, and ifnar1 mRNA was amplified by real-time RT-PCR. (D) Splenocytes from WT, Ifnar1−/−, and IFNAR1Texcl mice were stained for CD3, CD19, and IFNAR1. (E and F) Splenocytes were stimulated by IFN-α/β (250 U/ml) for 30 min (E) or for the indicated time (F) and then analyzed by Western blot. STAT1 and p-STAT1, 89/91 kDa; ISG15, 15 kDa; actin, 42 kDa. The images were spliced and joined, as indicated by white lines for the sake of the presentation. (G) Thymocytes were incubated with anti-IFNAR1-neutralizing antibody before IFN-α/β treatment and stained with a p-STAT1 antibody. The histogram plots show the p-STAT1-positive cells in the total live-cell gate (IFN treatment, bold, black line; antibody + IFN treatment, black line; no treatment, dotted line).

    Journal: Journal of Leukocyte Biology

    Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

    doi: 10.1189/jlb.3A1214-598R

    Figure Lengend Snippet: (A) Construct used to generate cd2–ifnar1 transgenic mice. (B) RT-PCR demonstrated lymphoid tissue-specific expression of transgenic ifnar1. With the use of a reverse primer for FLAG, this PCR reaction amplified the ifnar1 transcript only from transgenic mice. Br, Brain; Liv, liver; Lu, lung; Sp, spleen; Th, thymus. (C) RNA was extracted from CD3+-enriched T cells, and ifnar1 mRNA was amplified by real-time RT-PCR. (D) Splenocytes from WT, Ifnar1−/−, and IFNAR1Texcl mice were stained for CD3, CD19, and IFNAR1. (E and F) Splenocytes were stimulated by IFN-α/β (250 U/ml) for 30 min (E) or for the indicated time (F) and then analyzed by Western blot. STAT1 and p-STAT1, 89/91 kDa; ISG15, 15 kDa; actin, 42 kDa. The images were spliced and joined, as indicated by white lines for the sake of the presentation. (G) Thymocytes were incubated with anti-IFNAR1-neutralizing antibody before IFN-α/β treatment and stained with a p-STAT1 antibody. The histogram plots show the p-STAT1-positive cells in the total live-cell gate (IFN treatment, bold, black line; antibody + IFN treatment, black line; no treatment, dotted line).

    Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

    Techniques: Construct, Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Quantitative RT-PCR, Staining, Western Blot, Incubation

    (A–C) Splenocytes (A), peritoneal macrophages (B), and LN cells (C) were isolated from WT, Ifnar1−/−, and IFNAR1Texcl mice. Cells were treated with IFN-α/β (250 U/ml) for 15 min and double stained for CD3, CD4, CD8, CD11b, CD19, and p-STAT1. Dot plots show the double-positive cells in the total live-cell gate, and a representative dot plot/group is shown. (D and E) Thymidine uptake after anti (a)-CD3/anti-CD28 stimulation of splenocytes (D) and LN cells (E). Results shown are presented as mean values ± sem. (F) Cytokine production in splenocyte supernatants following stimulation with anti-CD3/anti-CD28. (G) Intracellular staining of splenocytes for IL-2 and IFN-γ after stimulation with anti-CD3/anti-CD28, followed by PMA/ionomycin/brefeldin A incubation. (H and I) CD3+-enriched T cells were stimulated under Th1- and Th17-polarizing conditions and stained for IFN-γ (H) and IL-17A (I). The bars represent the double-positive (CD4+ cytokine+) cells in the total live-cell gate. All data shown are representative of at least 2 independent experiments (n = 5 mice/genotype/experiment).

    Journal: Journal of Leukocyte Biology

    Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

    doi: 10.1189/jlb.3A1214-598R

    Figure Lengend Snippet: (A–C) Splenocytes (A), peritoneal macrophages (B), and LN cells (C) were isolated from WT, Ifnar1−/−, and IFNAR1Texcl mice. Cells were treated with IFN-α/β (250 U/ml) for 15 min and double stained for CD3, CD4, CD8, CD11b, CD19, and p-STAT1. Dot plots show the double-positive cells in the total live-cell gate, and a representative dot plot/group is shown. (D and E) Thymidine uptake after anti (a)-CD3/anti-CD28 stimulation of splenocytes (D) and LN cells (E). Results shown are presented as mean values ± sem. (F) Cytokine production in splenocyte supernatants following stimulation with anti-CD3/anti-CD28. (G) Intracellular staining of splenocytes for IL-2 and IFN-γ after stimulation with anti-CD3/anti-CD28, followed by PMA/ionomycin/brefeldin A incubation. (H and I) CD3+-enriched T cells were stimulated under Th1- and Th17-polarizing conditions and stained for IFN-γ (H) and IL-17A (I). The bars represent the double-positive (CD4+ cytokine+) cells in the total live-cell gate. All data shown are representative of at least 2 independent experiments (n = 5 mice/genotype/experiment).

    Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

    Techniques: Isolation, Staining, Incubation

    (A) Mean clinical scores for WT, Ifnar1−/−, and IFNAR1Texcl mice (n = 9 mice/genotype) after immunization with MOG35–55 peptide. Results shown are representative of 3 independent experiments and are presented as mean values ± sem. (B) Mean body weight of WT (n = 30), Ifnar1−/− (n = 28), and IFNAR1Texcl (n = 32) mice after EAE induction, pooled from 3 independent experiments. Results shown are presented as mean values ± sem.*P < 0.05, **P < 0.01: IFNAR1Texcl versus WT mice; and +P < 0.05, ++P < 0.01: IFNAR1Texcl versus Ifnar1−/− mice.

    Journal: Journal of Leukocyte Biology

    Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

    doi: 10.1189/jlb.3A1214-598R

    Figure Lengend Snippet: (A) Mean clinical scores for WT, Ifnar1−/−, and IFNAR1Texcl mice (n = 9 mice/genotype) after immunization with MOG35–55 peptide. Results shown are representative of 3 independent experiments and are presented as mean values ± sem. (B) Mean body weight of WT (n = 30), Ifnar1−/− (n = 28), and IFNAR1Texcl (n = 32) mice after EAE induction, pooled from 3 independent experiments. Results shown are presented as mean values ± sem.*P < 0.05, **P < 0.01: IFNAR1Texcl versus WT mice; and +P < 0.05, ++P < 0.01: IFNAR1Texcl versus Ifnar1−/− mice.

    Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

    Techniques:

    Clinical severity, day of onset, and peak of MOG 35–55 -induced EAE in IFNAR1 Texcl and control mice

    Journal: Journal of Leukocyte Biology

    Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

    doi: 10.1189/jlb.3A1214-598R

    Figure Lengend Snippet: Clinical severity, day of onset, and peak of MOG 35–55 -induced EAE in IFNAR1 Texcl and control mice

    Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

    Techniques: Control

    Clinical severity of WT and IFNAR1 Texcl mice upon PBS or IFN-β administration

    Journal: Journal of Leukocyte Biology

    Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

    doi: 10.1189/jlb.3A1214-598R

    Figure Lengend Snippet: Clinical severity of WT and IFNAR1 Texcl mice upon PBS or IFN-β administration

    Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

    Techniques:

    (A–D) Spinal cord sections from WT, Ifnar1−/−, and IFNAR1Texcl mice (n = 3 mice/genotype) were prepared 17 d upon EAE induction and stained with H&E (A), Luxol fast blue (B), and Bielschowsky's silver stain (C). Immunohistochemistry for CD3+ T cells was also performed (D). Arrows indicate immune cell infiltration (A), demyelination (B), axonal damage (C), and CD3+ T cell infiltration in the CNS parenchyma. Scale bars, 1 mm (A–C); 200 μm (D).

    Journal: Journal of Leukocyte Biology

    Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

    doi: 10.1189/jlb.3A1214-598R

    Figure Lengend Snippet: (A–D) Spinal cord sections from WT, Ifnar1−/−, and IFNAR1Texcl mice (n = 3 mice/genotype) were prepared 17 d upon EAE induction and stained with H&E (A), Luxol fast blue (B), and Bielschowsky's silver stain (C). Immunohistochemistry for CD3+ T cells was also performed (D). Arrows indicate immune cell infiltration (A), demyelination (B), axonal damage (C), and CD3+ T cell infiltration in the CNS parenchyma. Scale bars, 1 mm (A–C); 200 μm (D).

    Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

    Techniques: Staining, Silver Staining, Immunohistochemistry

    Splenocytes were isolated from WT, Ifnar1−/−, and IFNAR1Texcl mice, 10 d upon EAE induction. (A) Cells were stimulated with PMA/ionomycin/brefeldin A, and the frequency of IL- 17A-producing CD4+ T cells in the spleen was determined. The dot plots and bars represent IL-17A+ cells gated on total splenocytes. A representative dot plot per genotype is shown. (B) Expression levels of IL-17A by CD4+ T cells were evaluated by measuring MFI from (A) dot plots. (C) Splenocytes from all groups were restimulated ex vivo with MOG35–55 for 72 h, and the levels of secreted IL-17A were measured. (D) Frequency of CCR6+ splenocytes was determined (gated on the CD4+ compartment). All results are shown as means ± sem (n = 5 mice/genotype) and are representative from 2 independent experiments with similar results. *P < 0.05, **P < 0.01, ***P < 0.001.

    Journal: Journal of Leukocyte Biology

    Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

    doi: 10.1189/jlb.3A1214-598R

    Figure Lengend Snippet: Splenocytes were isolated from WT, Ifnar1−/−, and IFNAR1Texcl mice, 10 d upon EAE induction. (A) Cells were stimulated with PMA/ionomycin/brefeldin A, and the frequency of IL- 17A-producing CD4+ T cells in the spleen was determined. The dot plots and bars represent IL-17A+ cells gated on total splenocytes. A representative dot plot per genotype is shown. (B) Expression levels of IL-17A by CD4+ T cells were evaluated by measuring MFI from (A) dot plots. (C) Splenocytes from all groups were restimulated ex vivo with MOG35–55 for 72 h, and the levels of secreted IL-17A were measured. (D) Frequency of CCR6+ splenocytes was determined (gated on the CD4+ compartment). All results are shown as means ± sem (n = 5 mice/genotype) and are representative from 2 independent experiments with similar results. *P < 0.05, **P < 0.01, ***P < 0.001.

    Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

    Techniques: Isolation, Expressing, Ex Vivo

    Microarray analysis was performed on RNA from splenic, CD3+-enriched T cells and spinal cords from WT, Ifnar1−/−, and IFNAR1Texcl mice on d 0, 10, and 17 relative to EAE induction. (A and B) Heat map depicting the relative expression of selected genes that are different in expression between IFNAR1Texcl and WT T cells (A) and spinal cord (B) samples and were common in 2 independent experiments. Table 4 shows the function and number of molecules/category, as assessed by DAVID microarray software. The levels of mRNA transcripts for selected genes were measured in T cells (C) and spinal cords (D–F) by quantitative RT-PCR by use of single samples from each group at each time point (d 0: n = 3–5 mice/genotype; d 10: n = 3–4 mice/genotype; d 17: n = 2–3 mice/genotype). The results shown are the means ± sem of samples from 1 representative of 2 independent EAE experiments with similar results.*P < 0.05, **P < 0.01, ***P < 0.001.

    Journal: Journal of Leukocyte Biology

    Article Title: IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

    doi: 10.1189/jlb.3A1214-598R

    Figure Lengend Snippet: Microarray analysis was performed on RNA from splenic, CD3+-enriched T cells and spinal cords from WT, Ifnar1−/−, and IFNAR1Texcl mice on d 0, 10, and 17 relative to EAE induction. (A and B) Heat map depicting the relative expression of selected genes that are different in expression between IFNAR1Texcl and WT T cells (A) and spinal cord (B) samples and were common in 2 independent experiments. Table 4 shows the function and number of molecules/category, as assessed by DAVID microarray software. The levels of mRNA transcripts for selected genes were measured in T cells (C) and spinal cords (D–F) by quantitative RT-PCR by use of single samples from each group at each time point (d 0: n = 3–5 mice/genotype; d 10: n = 3–4 mice/genotype; d 17: n = 2–3 mice/genotype). The results shown are the means ± sem of samples from 1 representative of 2 independent EAE experiments with similar results.*P < 0.05, **P < 0.01, ***P < 0.001.

    Article Snippet: For the neutralization assay, thymocytes were incubated with 3 μg/ml anti-IFNAR1 neutralizing antibody (MAR1-5A3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C, 5% CO 2 , before culture with rmIFN-β (500 U/ml), and p-STAT1 expression was measured by flow cytometry, as described above.

    Techniques: Microarray, Expressing, Software, Quantitative RT-PCR