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Lack of C1q affects the level of immunoglobulins IgA (A) , IgG1 (B) , <t>IgG2a</t> (C) , IgG2b (D) , IgG3 (E) , and IgM (F) in infected mice. Whole blood was collected from mice infected with 10 4 Borrelia burgdorferi ) at 7, 10, 21, and 28 days post-infection. Serum level of immunoglobulins was determined by the Bio-Plex system employing the Luminex multianalyte profiling technology. White circles refer to samples from C57BL/6 mice, whereas red squares represent data from C1qα −/− mice ( n = 5; * P
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1) Product Images from "The Classical Complement Pathway Is Required to Control Borrelia burgdorferi Levels During Experimental Infection"

Article Title: The Classical Complement Pathway Is Required to Control Borrelia burgdorferi Levels During Experimental Infection

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00959

Lack of C1q affects the level of immunoglobulins IgA (A) , IgG1 (B) , IgG2a (C) , IgG2b (D) , IgG3 (E) , and IgM (F) in infected mice. Whole blood was collected from mice infected with 10 4 Borrelia burgdorferi ) at 7, 10, 21, and 28 days post-infection. Serum level of immunoglobulins was determined by the Bio-Plex system employing the Luminex multianalyte profiling technology. White circles refer to samples from C57BL/6 mice, whereas red squares represent data from C1qα −/− mice ( n = 5; * P
Figure Legend Snippet: Lack of C1q affects the level of immunoglobulins IgA (A) , IgG1 (B) , IgG2a (C) , IgG2b (D) , IgG3 (E) , and IgM (F) in infected mice. Whole blood was collected from mice infected with 10 4 Borrelia burgdorferi ) at 7, 10, 21, and 28 days post-infection. Serum level of immunoglobulins was determined by the Bio-Plex system employing the Luminex multianalyte profiling technology. White circles refer to samples from C57BL/6 mice, whereas red squares represent data from C1qα −/− mice ( n = 5; * P

Techniques Used: Infection, Mouse Assay, Luminex

Lack of C1q alters the level of Borrelia -specific antibody subtypes IgM (A) , IgG1 (B) , IgG2a (C) , IgG2b (D) , and IgG3 (E) in infected mice. Whole blood was collected from mice infected with 10 4 Borrelia burgdorferi ) at 7, 10, 21, and 28 days post-infection. Serum level of Borrelia -specific immunoglobulin subtypes was determined by ELISA using total protein lysate derived from B. burgdorferi strain MSK5. White columns refer to C57BL/6 mice, whereas red columns represent data from C1qα −/− mice ( n = 5; * P
Figure Legend Snippet: Lack of C1q alters the level of Borrelia -specific antibody subtypes IgM (A) , IgG1 (B) , IgG2a (C) , IgG2b (D) , and IgG3 (E) in infected mice. Whole blood was collected from mice infected with 10 4 Borrelia burgdorferi ) at 7, 10, 21, and 28 days post-infection. Serum level of Borrelia -specific immunoglobulin subtypes was determined by ELISA using total protein lysate derived from B. burgdorferi strain MSK5. White columns refer to C57BL/6 mice, whereas red columns represent data from C1qα −/− mice ( n = 5; * P

Techniques Used: Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay

2) Product Images from "Cross-Reactivity of Schistosoma mansoni Cytosolic Superoxide Dismutase, a Protective Vaccine Candidate, with Host Superoxide Dismutase and Identification of Parasite-Specific B Epitopes "

Article Title: Cross-Reactivity of Schistosoma mansoni Cytosolic Superoxide Dismutase, a Protective Vaccine Candidate, with Host Superoxide Dismutase and Identification of Parasite-Specific B Epitopes

Journal: Infection and Immunity

doi: 10.1128/IAI.72.5.2635-2647.2004

B-epitope map of SmCT-SOD. (A) SmCT-SOD sequence (153 amino acids; in italics) and derived peptides. Overlapping and nonoverlapping peptides of the 20-mer (in bold) were synthesized. The inclusive amino acids (aa) of the peptides were as follows: aa 7 to 26 (Pep 1); aa 22 to 41 (Pep 2); aa 64 to 83 (Pep 3); aa 84 to 103 (Pep 4); aa 101 to 120 (Pep 5); aa 118 to 137 (Pep 6); aa 134 to 153 (Pep 7). Pep 1 could not be synthesized in four attempts. (B and C) Reactivity of IgG antibodies from pooled sera of mice immunized with 5 mg of SmCT-SOD-GST-encapsulated microspheres/mouse (Microspheres-1); equivalent amount of SmCT-SOD-GST microspheres capable of releasing 50 μg/100 μl/mouse (Microspheres-2); 50 μg of CT-SOD-GST emulsified in alum/100 μl/mouse (Alum); mouse anti-hSOD antibodies (anti-hSOD); and 5 mg of GST-encapsulated microspheres (GST Microspheres) against GST and SmCT-SOD antigens (5 μg/ml) and SmCT-SOD-derived peptides (2 μg/ml). The sera of group Microspheres-1 were diluted starting from 1:2,500. The line represents the cutoff value (mean OD + 3 SD) of preimmune sera.
Figure Legend Snippet: B-epitope map of SmCT-SOD. (A) SmCT-SOD sequence (153 amino acids; in italics) and derived peptides. Overlapping and nonoverlapping peptides of the 20-mer (in bold) were synthesized. The inclusive amino acids (aa) of the peptides were as follows: aa 7 to 26 (Pep 1); aa 22 to 41 (Pep 2); aa 64 to 83 (Pep 3); aa 84 to 103 (Pep 4); aa 101 to 120 (Pep 5); aa 118 to 137 (Pep 6); aa 134 to 153 (Pep 7). Pep 1 could not be synthesized in four attempts. (B and C) Reactivity of IgG antibodies from pooled sera of mice immunized with 5 mg of SmCT-SOD-GST-encapsulated microspheres/mouse (Microspheres-1); equivalent amount of SmCT-SOD-GST microspheres capable of releasing 50 μg/100 μl/mouse (Microspheres-2); 50 μg of CT-SOD-GST emulsified in alum/100 μl/mouse (Alum); mouse anti-hSOD antibodies (anti-hSOD); and 5 mg of GST-encapsulated microspheres (GST Microspheres) against GST and SmCT-SOD antigens (5 μg/ml) and SmCT-SOD-derived peptides (2 μg/ml). The sera of group Microspheres-1 were diluted starting from 1:2,500. The line represents the cutoff value (mean OD + 3 SD) of preimmune sera.

Techniques Used: Sequencing, Derivative Assay, Synthesized, Mouse Assay

Reactivity of antibody isotype against SmCT-SOD. Reactivity of IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 antibodies from pooled sera of mice immunized with equivalent amounts of SmCT-SOD-GST microspheres capable of releasing 50 μg/100 μl/mouse (CT-SOD microspheres), 50 μg of SmCT-SOD-GST mixed in alum/100 μl/mouse (CT-SOD Alum), and controls (GST microspheres only and GST alum only). The background reactivities to SmCT-SOD were subtracted (SmCT-SOD-GST − GST microspheres or GST alum). The line represents the cutoff value that was calculated using the mean + 3 SD of preimmune mouse sera absorbance at the lowest dilution (1:80).
Figure Legend Snippet: Reactivity of antibody isotype against SmCT-SOD. Reactivity of IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 antibodies from pooled sera of mice immunized with equivalent amounts of SmCT-SOD-GST microspheres capable of releasing 50 μg/100 μl/mouse (CT-SOD microspheres), 50 μg of SmCT-SOD-GST mixed in alum/100 μl/mouse (CT-SOD Alum), and controls (GST microspheres only and GST alum only). The background reactivities to SmCT-SOD were subtracted (SmCT-SOD-GST − GST microspheres or GST alum). The line represents the cutoff value that was calculated using the mean + 3 SD of preimmune mouse sera absorbance at the lowest dilution (1:80).

Techniques Used: Mouse Assay

(A and B) Comparison of anti-SmCT-SOD antibody responses to hSOD under denaturing (A) and nondenaturing (B) conditions. The top gels are acrylamide gels of SmCT-SOD-GST, GST, BSA, SmGPX, and hSOD antigens stained with Coomassie blue. The middle panels are Western blots after incubation with rabbit anti-SmCT-SOD-GST IgG antibodies (1:2,500). Bottom gels are Western blots after incubation with mouse anti-SmCT-SOD-GST IgG antibodies (1:10,000). (C) ELISA results showing the mean reactivity (± standard error of the mean) of rabbit anti-SmCT-SOD-GST sera (1:500) to SmCT-SOD and hSOD antigens from three experiments. (D) ELISA results showing reactivities of mouse anti-hSOD sera against SmCT-SOD-GST, GST, and hSOD proteins. The line represents the cutoff value (mean OD value + 3 times the SD) of preimmune sera.
Figure Legend Snippet: (A and B) Comparison of anti-SmCT-SOD antibody responses to hSOD under denaturing (A) and nondenaturing (B) conditions. The top gels are acrylamide gels of SmCT-SOD-GST, GST, BSA, SmGPX, and hSOD antigens stained with Coomassie blue. The middle panels are Western blots after incubation with rabbit anti-SmCT-SOD-GST IgG antibodies (1:2,500). Bottom gels are Western blots after incubation with mouse anti-SmCT-SOD-GST IgG antibodies (1:10,000). (C) ELISA results showing the mean reactivity (± standard error of the mean) of rabbit anti-SmCT-SOD-GST sera (1:500) to SmCT-SOD and hSOD antigens from three experiments. (D) ELISA results showing reactivities of mouse anti-hSOD sera against SmCT-SOD-GST, GST, and hSOD proteins. The line represents the cutoff value (mean OD value + 3 times the SD) of preimmune sera.

Techniques Used: Staining, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay

Reactivity of antisera against SmCT-SOD from immunization with different adjuvants. (A and B) Reactivity against SmCT-SOD-GST (A) and GST (B) recombinant antigens of IgG antibodies from pooled sera of mice preimmune, immunized, and postboost with 5 mg of PLA microspheres encapsulated with or without GST and SmCT-SOD-GST antigens/100 μl/mouse. (C and D) Reactivity of IgG antibodies from pooled sera of mice immunized twice with an equivalent amount of the PLA microspheres adjusted to release 50 μg of recombinant antigen/100 μl/mouse or prime-boosted with the same antigens mixed in alum (50 μg/100 μl) against SmCT-SOD (C) and GST (D). The background reactivities of control mice vaccinated with microspheres alone or alum alone were subtracted from the respective other group. A group of mice were prime-boosted with 50 μg of naked pcDNA encoding SmCT-SOD. Rabbit anti-SmCT-SOD was included as a positive control. The line represents the cutoff value that was calculated using the mean + 3 SD of preimmune mouse sera absorbance at the lowest dilution (1:80).
Figure Legend Snippet: Reactivity of antisera against SmCT-SOD from immunization with different adjuvants. (A and B) Reactivity against SmCT-SOD-GST (A) and GST (B) recombinant antigens of IgG antibodies from pooled sera of mice preimmune, immunized, and postboost with 5 mg of PLA microspheres encapsulated with or without GST and SmCT-SOD-GST antigens/100 μl/mouse. (C and D) Reactivity of IgG antibodies from pooled sera of mice immunized twice with an equivalent amount of the PLA microspheres adjusted to release 50 μg of recombinant antigen/100 μl/mouse or prime-boosted with the same antigens mixed in alum (50 μg/100 μl) against SmCT-SOD (C) and GST (D). The background reactivities of control mice vaccinated with microspheres alone or alum alone were subtracted from the respective other group. A group of mice were prime-boosted with 50 μg of naked pcDNA encoding SmCT-SOD. Rabbit anti-SmCT-SOD was included as a positive control. The line represents the cutoff value that was calculated using the mean + 3 SD of preimmune mouse sera absorbance at the lowest dilution (1:80).

Techniques Used: Recombinant, Mouse Assay, Proximity Ligation Assay, Positive Control

Isotype responses to SmCT-SOD-derived peptides. Reactivity of IgG1 (A), IgG2a (B), IgG2b (C), and IgG3 (D) antibodies from pooled sera of mice immunized with 5 mg of SmCT-SOD-GST-encapsulated microspheres/mouse (Microspheres-1); equivalent amount of SmCT-SOD-GST microspheres capable of releasing 50 μg/100 μl/mouse (Microspheres-2); 50 μg of CT-SOD-GST mixed in alum/100 μl/mouse (Alum); 100 μg of SmCT-SOD DNA/100 μl/mouse (DNA) against SmCT-SOD antigens (2 μg/ml) and SmCT-SOD derived-peptides (2 μg/ml). The sera of the Microspheres-1 group were diluted starting from 1:2,500. The line represents the cutoff value (mean OD + 3 SD) of preimmune sera.
Figure Legend Snippet: Isotype responses to SmCT-SOD-derived peptides. Reactivity of IgG1 (A), IgG2a (B), IgG2b (C), and IgG3 (D) antibodies from pooled sera of mice immunized with 5 mg of SmCT-SOD-GST-encapsulated microspheres/mouse (Microspheres-1); equivalent amount of SmCT-SOD-GST microspheres capable of releasing 50 μg/100 μl/mouse (Microspheres-2); 50 μg of CT-SOD-GST mixed in alum/100 μl/mouse (Alum); 100 μg of SmCT-SOD DNA/100 μl/mouse (DNA) against SmCT-SOD antigens (2 μg/ml) and SmCT-SOD derived-peptides (2 μg/ml). The sera of the Microspheres-1 group were diluted starting from 1:2,500. The line represents the cutoff value (mean OD + 3 SD) of preimmune sera.

Techniques Used: Derivative Assay, Mouse Assay

3) Product Images from "The Schistosoma mansoni Hepatic Egg Granuloma Provides a Favorable Microenvironment for Sustained Growth of Leishmania donovani"

Article Title: The Schistosoma mansoni Hepatic Egg Granuloma Provides a Favorable Microenvironment for Sustained Growth of Leishmania donovani

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2006.051319

Preferential generation of L. donovani foci within the schistosome egg granulomatous tissue. A: Liver sections from S/L mice at +8 weeks after co-infection were stained for L. donovani amastigotes using immune hamster anti- L. donovani serum and Alexa Fluor 546 goat anti-hamster IgG (red, arrow , AM) and for mannose receptor (MR) with anti-CD 206 and Alexa Fluor 488 goat anti-rat IgG (green). To ensure staining specificity, liver sections from the same S/L mice were stained with isotype control rat IgG2a (control for the MR staining) or normal hamster serum (control for the amastigote staining). No nonspecific staining was seen. Strong specific MR staining can be seen in the granulomatous tissue surrounding the S. mansoni eggs ( arrow , EG). The parenchymal Küpffer cells stain weakly for MR ( arrow , KC), and MR-positive cells are absent from the granulomas surrounding L. donovani amastigotes in the parenchyma ( inset ). A higher density of red-staining L. donovani amastigotes can be seen in foci of L. donovani infection in the periphery of the S. mansoni egg granulomas compared with inside the L. donovani granulomas (LG) in the parenchymal tissue. B: This was confirmed quantitatively in H E-stained tissue sections by counting the density of L. donovani foci in the egg granulomatous tissue of S/L mice (S/L G, open square) compared with the surrounding parenchyma (S/L P, filled square) and the parenchyma of L mice (L, open circle). For each mouse 50 adjacent fields were counted at ×400 magnification. The data in B show the mean ± SE from five mice per group and is representative of two separate experiments. * P
Figure Legend Snippet: Preferential generation of L. donovani foci within the schistosome egg granulomatous tissue. A: Liver sections from S/L mice at +8 weeks after co-infection were stained for L. donovani amastigotes using immune hamster anti- L. donovani serum and Alexa Fluor 546 goat anti-hamster IgG (red, arrow , AM) and for mannose receptor (MR) with anti-CD 206 and Alexa Fluor 488 goat anti-rat IgG (green). To ensure staining specificity, liver sections from the same S/L mice were stained with isotype control rat IgG2a (control for the MR staining) or normal hamster serum (control for the amastigote staining). No nonspecific staining was seen. Strong specific MR staining can be seen in the granulomatous tissue surrounding the S. mansoni eggs ( arrow , EG). The parenchymal Küpffer cells stain weakly for MR ( arrow , KC), and MR-positive cells are absent from the granulomas surrounding L. donovani amastigotes in the parenchyma ( inset ). A higher density of red-staining L. donovani amastigotes can be seen in foci of L. donovani infection in the periphery of the S. mansoni egg granulomas compared with inside the L. donovani granulomas (LG) in the parenchymal tissue. B: This was confirmed quantitatively in H E-stained tissue sections by counting the density of L. donovani foci in the egg granulomatous tissue of S/L mice (S/L G, open square) compared with the surrounding parenchyma (S/L P, filled square) and the parenchyma of L mice (L, open circle). For each mouse 50 adjacent fields were counted at ×400 magnification. The data in B show the mean ± SE from five mice per group and is representative of two separate experiments. * P

Techniques Used: Mouse Assay, Infection, Staining

4) Product Images from "Trypanosoma cruzi infection induces a massive extrafollicular and follicular splenic B-cell response which is a high source of non-parasite-specific antibodies"

Article Title: Trypanosoma cruzi infection induces a massive extrafollicular and follicular splenic B-cell response which is a high source of non-parasite-specific antibodies

Journal: Immunology

doi: 10.1111/j.1365-2567.2010.03347.x

Parasite-specific antibodies in sera from Trypanosoma cruzi -infected mice. T. cruzi -specific IgM and IgG isotypes titres were determined by ELISA in sera from normal (day 0) or T. cruzi -infected mice at different days post-infection. Test sera were considered
Figure Legend Snippet: Parasite-specific antibodies in sera from Trypanosoma cruzi -infected mice. T. cruzi -specific IgM and IgG isotypes titres were determined by ELISA in sera from normal (day 0) or T. cruzi -infected mice at different days post-infection. Test sera were considered

Techniques Used: Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay

Immunoglobulin production by spleen cells from Trypanosoma cruzi -infected mice. IgM and IgG isotype concentrations, determined by ELISA, in culture supernatant of splenic cells obtained from normal (day 0) or T. cruzi -infected mice at different days post-infection.
Figure Legend Snippet: Immunoglobulin production by spleen cells from Trypanosoma cruzi -infected mice. IgM and IgG isotype concentrations, determined by ELISA, in culture supernatant of splenic cells obtained from normal (day 0) or T. cruzi -infected mice at different days post-infection.

Techniques Used: Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay

5) Product Images from "CD4+ T Lymphocytes Are Critical Mediators of Tumor Immunity to Simian Virus 40 Large Tumor Antigen Induced by Vaccination with Plasmid DNA ▿"

Article Title: CD4+ T Lymphocytes Are Critical Mediators of Tumor Immunity to Simian Virus 40 Large Tumor Antigen Induced by Vaccination with Plasmid DNA ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00543-11

Immunization with pCVM-Tag induces a mixed IgG subtype distribution of antibodies to SV40 Tag.
Figure Legend Snippet: Immunization with pCVM-Tag induces a mixed IgG subtype distribution of antibodies to SV40 Tag.

Techniques Used:

6) Product Images from "Regulatory T Cells in the Control of Autoimmunity: the Essential Role of Transforming Growth Factor ? and Interleukin 4 in the Prevention of Autoimmune Thyroiditis in Rats by Peripheral CD4+CD45RC− Cells and CD4+CD8− Thymocytes "

Article Title: Regulatory T Cells in the Control of Autoimmunity: the Essential Role of Transforming Growth Factor ? and Interleukin 4 in the Prevention of Autoimmune Thyroiditis in Rats by Peripheral CD4+CD45RC− Cells and CD4+CD8− Thymocytes

Journal: The Journal of Experimental Medicine

doi:

Treatment of TxX rats with neutralizing mAbs against TGF-β or IL-4 abrogates protection from thyroiditis by both CD4 + CD45RC − cells and CD4 + CD8 − thymocytes. Shortly after their last irradiation, groups of TxX PVG rats were injected with either 5 × 10 6 CD4 + CD45RC − cells purified from TDLs of 12-wk-old normal PVG rats or 10 6 CD4 + CD8 − thymocytes purified from thymus of 6-wk-old normal PVG rats. Control rats received no cells. Rats reconstituted with cells were treated the day before and the day after cell transfer and then twice weekly for 4 wk with either the anti–TGF-β–neutralizing mAb 2G.7 (mouse IgG2a; 2 mg/injection) or the anti–IL-4–neutralizing mAb OX81 (mouse IgG1; 2 mg/injection). Control rats reconstituted with cells either received no further treatment or were injected with one of the isotype-control mAbs OX21 (mouse IgG1; 2 mg/injection) or W6/32 (mouse IgG2a; 2 mg/injection) specific for human determinants. Data represent peak anti-Tg antibody levels of individual TxX rats reconstituted with either CD4 + CD45RC − cells (A) or CD4 + CD8 − thymocytes (B). Anti-Tg titers are expressed as percentage of standard and data are the pool of five independent experiments. Statistics versus controls: a P
Figure Legend Snippet: Treatment of TxX rats with neutralizing mAbs against TGF-β or IL-4 abrogates protection from thyroiditis by both CD4 + CD45RC − cells and CD4 + CD8 − thymocytes. Shortly after their last irradiation, groups of TxX PVG rats were injected with either 5 × 10 6 CD4 + CD45RC − cells purified from TDLs of 12-wk-old normal PVG rats or 10 6 CD4 + CD8 − thymocytes purified from thymus of 6-wk-old normal PVG rats. Control rats received no cells. Rats reconstituted with cells were treated the day before and the day after cell transfer and then twice weekly for 4 wk with either the anti–TGF-β–neutralizing mAb 2G.7 (mouse IgG2a; 2 mg/injection) or the anti–IL-4–neutralizing mAb OX81 (mouse IgG1; 2 mg/injection). Control rats reconstituted with cells either received no further treatment or were injected with one of the isotype-control mAbs OX21 (mouse IgG1; 2 mg/injection) or W6/32 (mouse IgG2a; 2 mg/injection) specific for human determinants. Data represent peak anti-Tg antibody levels of individual TxX rats reconstituted with either CD4 + CD45RC − cells (A) or CD4 + CD8 − thymocytes (B). Anti-Tg titers are expressed as percentage of standard and data are the pool of five independent experiments. Statistics versus controls: a P

Techniques Used: Irradiation, Injection, Purification

Prevention of thyroiditis development in TxX PVG rats by their reconstitution with either CD4 + CD45RC − T cells or CD4 + CD8 − thymocytes. Unfractionated CD4 + and CD4 + CD45RC − cells were purified from TDLs of 12-wk-old normal PVG rats by Dynal bead depletion. CD4 + CD45RC + cells were purified from CD4 + TDLs by positive selection using MACS beads, whereas CD4 + CD8 − thymocytes were purified from thymus of 6-wk-old rats by depletion of CD8 + cells as described in Materials and Methods. Shortly after their last irradiation, TxX PVG rats were reconstituted with 10 7 of unfractionated CD4 + cells, CD4 + CD45RC − cells, CD4 + CD45RC + cells, or CD4 + CD8 − thymocytes, while control rats received no cells. Data represent peak anti-Tg IgG antibody levels in individual TxX rats from groups reconstituted with different T cell subsets. Anti-Tg titers are expressed as percentage of a standard and data are the pool of six independent experiments. Statistics versus controls: a P
Figure Legend Snippet: Prevention of thyroiditis development in TxX PVG rats by their reconstitution with either CD4 + CD45RC − T cells or CD4 + CD8 − thymocytes. Unfractionated CD4 + and CD4 + CD45RC − cells were purified from TDLs of 12-wk-old normal PVG rats by Dynal bead depletion. CD4 + CD45RC + cells were purified from CD4 + TDLs by positive selection using MACS beads, whereas CD4 + CD8 − thymocytes were purified from thymus of 6-wk-old rats by depletion of CD8 + cells as described in Materials and Methods. Shortly after their last irradiation, TxX PVG rats were reconstituted with 10 7 of unfractionated CD4 + cells, CD4 + CD45RC − cells, CD4 + CD45RC + cells, or CD4 + CD8 − thymocytes, while control rats received no cells. Data represent peak anti-Tg IgG antibody levels in individual TxX rats from groups reconstituted with different T cell subsets. Anti-Tg titers are expressed as percentage of a standard and data are the pool of six independent experiments. Statistics versus controls: a P

Techniques Used: Purification, Selection, Magnetic Cell Separation, Irradiation

IgG isotypes of anti-Tg antibodies in TxX rats is not affected by their treatment with anti–IL-4 or anti–TGF-β blocking mAbs. The relative isotype usage of anti-Tg IgG responses was determined for TxX rats reconstituted with CD4 + CD45RC − cells but treated with either anti–IL-4 ( n = 9) or anti–TGF-β ( n = 7) blocking mAbs and those reconstituted with CD4 + CD8 − thymocytes and treated with either anti-IL-4 ( n = 6) or anti-TGF-β ( n = 9) blocking mAbs from the experiments described in Fig. 5 . The isotype of anti-Tg IgG in sera of control rats ( n = 16) from both these series of experiments was similarly determined by specific ELISA. Data are expressed as the mean percentage of the anti-Tg response for a given IgG isotype where 100% is the sum of the ODs for individual isotypes above background of normal PVG sera in 1:10 dilutions of an experimental serum. Error bars indicate SD.
Figure Legend Snippet: IgG isotypes of anti-Tg antibodies in TxX rats is not affected by their treatment with anti–IL-4 or anti–TGF-β blocking mAbs. The relative isotype usage of anti-Tg IgG responses was determined for TxX rats reconstituted with CD4 + CD45RC − cells but treated with either anti–IL-4 ( n = 9) or anti–TGF-β ( n = 7) blocking mAbs and those reconstituted with CD4 + CD8 − thymocytes and treated with either anti-IL-4 ( n = 6) or anti-TGF-β ( n = 9) blocking mAbs from the experiments described in Fig. 5 . The isotype of anti-Tg IgG in sera of control rats ( n = 16) from both these series of experiments was similarly determined by specific ELISA. Data are expressed as the mean percentage of the anti-Tg response for a given IgG isotype where 100% is the sum of the ODs for individual isotypes above background of normal PVG sera in 1:10 dilutions of an experimental serum. Error bars indicate SD.

Techniques Used: Blocking Assay, Enzyme-linked Immunosorbent Assay

Thyroiditis in PVG rats after their thymectomy and irradiation develops independently of CD8 + cells and is characterized by a Th2-like anti-Tg IgG response. Female PVG rats were thymectomized at 3 wk of age followed 1 wk later, by four doses of 275 rad 137 Cs γ-irradiation at 2-wk intervals. (A) The isotype of anti-Tg IgG antibodies in sera of TxX rats with thyroiditis ( n = 25) and normal 12-wk-old female PVG rats immunized with Tg (50 μg/rat) in CFA ( n = 5) was determined by specific ELISA. Data are expressed as the mean percentage of the anti-Tg response for each IgG isotype where 100% is the sum of the ODs for individual isotypes above background of normal PVG sera in 1:10 dilutions of an experimental serum. Error bars indicate SD. (B) The requirement for CD8 + cells in the development of thyroiditis in TxX PVG rats was determined by their injection at the time of thymectomy and 7 d later with either the anti-CD8-depleting mAb OX8 (0.5 mg/injection) or PBS as control. Development of anti-Tg IgG responses was monitored between 4 and 12 wk after the last irradiation by specific ELISA. Data represent peak anti-Tg IgG titers expressed as percentage of standard for individual TxX rats. FACS ® analysis of splenocytes from OX8-treated rats, 12 wk after the last irradiation, showed that
Figure Legend Snippet: Thyroiditis in PVG rats after their thymectomy and irradiation develops independently of CD8 + cells and is characterized by a Th2-like anti-Tg IgG response. Female PVG rats were thymectomized at 3 wk of age followed 1 wk later, by four doses of 275 rad 137 Cs γ-irradiation at 2-wk intervals. (A) The isotype of anti-Tg IgG antibodies in sera of TxX rats with thyroiditis ( n = 25) and normal 12-wk-old female PVG rats immunized with Tg (50 μg/rat) in CFA ( n = 5) was determined by specific ELISA. Data are expressed as the mean percentage of the anti-Tg response for each IgG isotype where 100% is the sum of the ODs for individual isotypes above background of normal PVG sera in 1:10 dilutions of an experimental serum. Error bars indicate SD. (B) The requirement for CD8 + cells in the development of thyroiditis in TxX PVG rats was determined by their injection at the time of thymectomy and 7 d later with either the anti-CD8-depleting mAb OX8 (0.5 mg/injection) or PBS as control. Development of anti-Tg IgG responses was monitored between 4 and 12 wk after the last irradiation by specific ELISA. Data represent peak anti-Tg IgG titers expressed as percentage of standard for individual TxX rats. FACS ® analysis of splenocytes from OX8-treated rats, 12 wk after the last irradiation, showed that

Techniques Used: Irradiation, Enzyme-linked Immunosorbent Assay, Injection, FACS

7) Product Images from "Thymic stromal lymphopoietin (TSLP) acts as a potent mucosal adjuvant for HIV-1 gp140 vaccination in mice"

Article Title: Thymic stromal lymphopoietin (TSLP) acts as a potent mucosal adjuvant for HIV-1 gp140 vaccination in mice

Journal: European Journal of Immunology

doi: 10.1002/eji.201141787

TSLP induces gp140-specific immune responses after intranasal immunisation. Mice were immunised with 10 μg gp140 in the presence or absence of 5 μg TSLP, APRIL or BAFF i.n. three times at 3 week intervals. Anti-gp140 IgA (left) and IgG (right) were measured 3 weeks after the final immunisation by ELISA in (A) serum and (B) vaginal lavage. Data are shown as endpoint titres, with lines representing geometric means of n =5 mice; data are representative of two experiments. * p
Figure Legend Snippet: TSLP induces gp140-specific immune responses after intranasal immunisation. Mice were immunised with 10 μg gp140 in the presence or absence of 5 μg TSLP, APRIL or BAFF i.n. three times at 3 week intervals. Anti-gp140 IgA (left) and IgG (right) were measured 3 weeks after the final immunisation by ELISA in (A) serum and (B) vaginal lavage. Data are shown as endpoint titres, with lines representing geometric means of n =5 mice; data are representative of two experiments. * p

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

TSLP shifts the immune response to gp140 to Th2 Mice were immunised three times i.n. with 10 μg gp140 alone or with 5 μg TSLP or CT. (A) Anti-gp140 IgG 1 and IgG 2a and (B) IgE were measured in serum by ELISA. (C) Splenocytes were stimulated with a CN54 gp140 peptide pool and specific cytokine production was measured in CD4 + T cells by flow cytometry. (D) Mice were immunised twice at 2 week intervals, followed by three daily i.n. challenges with 20 μg gp140 in 100 μL and were culled 3 days later. (E) gp140-specific IgE was measured in sera. (F) Cells were collected from bronchoalveolar lavage (BAL) and counted and (G) differential cell counts performed. Representative PAS-stained sections of lungs from (H) PBS, (I) gp140, (J) gp140:TSLP, (K) gp140:CT, (L) IP gp140:alum. Data are shown as mean of n =5 mice+SD, experiment performed once. * p
Figure Legend Snippet: TSLP shifts the immune response to gp140 to Th2 Mice were immunised three times i.n. with 10 μg gp140 alone or with 5 μg TSLP or CT. (A) Anti-gp140 IgG 1 and IgG 2a and (B) IgE were measured in serum by ELISA. (C) Splenocytes were stimulated with a CN54 gp140 peptide pool and specific cytokine production was measured in CD4 + T cells by flow cytometry. (D) Mice were immunised twice at 2 week intervals, followed by three daily i.n. challenges with 20 μg gp140 in 100 μL and were culled 3 days later. (E) gp140-specific IgE was measured in sera. (F) Cells were collected from bronchoalveolar lavage (BAL) and counted and (G) differential cell counts performed. Representative PAS-stained sections of lungs from (H) PBS, (I) gp140, (J) gp140:TSLP, (K) gp140:CT, (L) IP gp140:alum. Data are shown as mean of n =5 mice+SD, experiment performed once. * p

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Staining

TSLP induces similar humoral but not cellular responses to CT. Mice were immunised three times i.n. with 10 μg gp140 alone (+),with 5 μg TSLP (△) or CT (◊). Anti-gp140 IgA and IgG were measured by ELISA in (A, B) serum, (C, D) vaginal lavage and (E, F) faecal extracts obtained from mice 3 weeks after the final immunisation. (G) Anti-gp140 IgA and IgG ASC were measured by B-cell ELISPOT in spleen from mice 3 wks after the final immunisation. (H) 10 days after the final immunisation, splenocytes were isolated and cultured for 5 days with CN54 gp140 protein and proliferation measured by 3 H thymidine incorporation. Data points represent single mice, bars represent mean of n =5 mice+SD (G, H). Data are representative of two experiments. * p
Figure Legend Snippet: TSLP induces similar humoral but not cellular responses to CT. Mice were immunised three times i.n. with 10 μg gp140 alone (+),with 5 μg TSLP (△) or CT (◊). Anti-gp140 IgA and IgG were measured by ELISA in (A, B) serum, (C, D) vaginal lavage and (E, F) faecal extracts obtained from mice 3 weeks after the final immunisation. (G) Anti-gp140 IgA and IgG ASC were measured by B-cell ELISPOT in spleen from mice 3 wks after the final immunisation. (H) 10 days after the final immunisation, splenocytes were isolated and cultured for 5 days with CN54 gp140 protein and proliferation measured by 3 H thymidine incorporation. Data points represent single mice, bars represent mean of n =5 mice+SD (G, H). Data are representative of two experiments. * p

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Isolation, Cell Culture

TSLP induces sustained humoral and cellular immune responses to gp140. Mice were immunised four times i.n. with 10 μg gp140 alone (▪) or with 5 μg TSLP (○) or CT (◊). The fourth immunisation was given 6 months after the third immunisation. Anti-gp140 IgA and IgG were measured by ELISA in (A, B) serum and (C, D) vaginal lavage before every immunisation and 3 weeks after the final immunisation. (E) CN54 gp140 IgA and IgG ASC were measured by B-cell ELISPOT in spleens from mice 3 weeks after the final immunisation. Antigen-specific T-cell proliferation of splenocytes was assessed after in vitro stimulation with gp140 after 120 h using (F) 3 H-thymidine and (G) CFSE dilution. Data are shown as mean of n =5 mice+SD, experiment was performed once. * p
Figure Legend Snippet: TSLP induces sustained humoral and cellular immune responses to gp140. Mice were immunised four times i.n. with 10 μg gp140 alone (▪) or with 5 μg TSLP (○) or CT (◊). The fourth immunisation was given 6 months after the third immunisation. Anti-gp140 IgA and IgG were measured by ELISA in (A, B) serum and (C, D) vaginal lavage before every immunisation and 3 weeks after the final immunisation. (E) CN54 gp140 IgA and IgG ASC were measured by B-cell ELISPOT in spleens from mice 3 weeks after the final immunisation. Antigen-specific T-cell proliferation of splenocytes was assessed after in vitro stimulation with gp140 after 120 h using (F) 3 H-thymidine and (G) CFSE dilution. Data are shown as mean of n =5 mice+SD, experiment was performed once. * p

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, In Vitro

I.n. immunisation induces higher gp140-specific IgA responses than i.d immunisation. Mice were immunised i.n. (open symbols) or i.d. (black symbols) three times with 10 μg gp140 alone or with 5 μg TSLP or CT. (A, C) Anti-gp140 IgG and (B, D) anti-gp140 IgA were measured by ELISA (A, B) in serum 3 weeks after priming (first immunisation), first boost (second immunisation) and final immunisation (third immunisation) and (C, D) in vaginal lavage, faecal extracts and nasal lavage 3 weeks after the final immunisation. Data are shown as endpoint titres, with lines representing geometric means of n =5 mice; data are representative of two experiments. *** p
Figure Legend Snippet: I.n. immunisation induces higher gp140-specific IgA responses than i.d immunisation. Mice were immunised i.n. (open symbols) or i.d. (black symbols) three times with 10 μg gp140 alone or with 5 μg TSLP or CT. (A, C) Anti-gp140 IgG and (B, D) anti-gp140 IgA were measured by ELISA (A, B) in serum 3 weeks after priming (first immunisation), first boost (second immunisation) and final immunisation (third immunisation) and (C, D) in vaginal lavage, faecal extracts and nasal lavage 3 weeks after the final immunisation. Data are shown as endpoint titres, with lines representing geometric means of n =5 mice; data are representative of two experiments. *** p

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

8) Product Images from "Epicutaneous Immunotherapy (EPIT) Blocks the Allergic Esophago-Gastro-Enteropathy Induced by Sustained Oral Exposure to Peanuts in Sensitized Mice"

Article Title: Epicutaneous Immunotherapy (EPIT) Blocks the Allergic Esophago-Gastro-Enteropathy Induced by Sustained Oral Exposure to Peanuts in Sensitized Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0031967

Study design for induction of eosinophilic esophagatis and enteropathy and for the effect of EPIT on the induction of digestive lesions. (A) Fourty mice were sensitized to peanut proteins in the first phase. Then a resting period with no treatment and no peanut administration was applied. After that, a peanut regimen for 10 days was given to sensitized and naïve mice (n = 40). Mice were then sacrificed to analyze esophagus and jejunum samples by histology and RT-qPCR. (B) Twenty mice were sensitized to peanut proteins in the first phase. Epicutaneous immunotherapy was conducted for 8 weeks in 1à sensitized mice (EPIT) and 10 other sensitized mice received a Sham treatment (Sham). After a sustained oral challenge, mice were sacrificed to analyze esophagus and jejunum samples by histology and RT-qPCR. Blood samples were taken every 2 weeks to measure specific immunoglobulins (IgE, IgG1, IgG2a).
Figure Legend Snippet: Study design for induction of eosinophilic esophagatis and enteropathy and for the effect of EPIT on the induction of digestive lesions. (A) Fourty mice were sensitized to peanut proteins in the first phase. Then a resting period with no treatment and no peanut administration was applied. After that, a peanut regimen for 10 days was given to sensitized and naïve mice (n = 40). Mice were then sacrificed to analyze esophagus and jejunum samples by histology and RT-qPCR. (B) Twenty mice were sensitized to peanut proteins in the first phase. Epicutaneous immunotherapy was conducted for 8 weeks in 1à sensitized mice (EPIT) and 10 other sensitized mice received a Sham treatment (Sham). After a sustained oral challenge, mice were sacrificed to analyze esophagus and jejunum samples by histology and RT-qPCR. Blood samples were taken every 2 weeks to measure specific immunoglobulins (IgE, IgG1, IgG2a).

Techniques Used: Mouse Assay, Quantitative RT-PCR

Effect of EPIT on systemic response induced in mice after oral sensitization. (A) Quantity of specific IgE expressed in µg.ml −1 for each group. (B) Quantity of specific IgG2a expressed in µg.ml −1 for each group. (C) Determination of the IgG1/IgG2a ratio expressed for each group. D44 (week 6) concords with the end of sensitization and from D44 to D99 (weeks 7 to 14) with the immunotherapy. Data are expressed as means ± SD for each group of 10 mice. (D–H) Measurement of Th2 cytokine levels (IL-4, IL-5, IL-10, IL-13) and IFN-γ secretion by splenocytes collected from each group of mice (EPIT, Sham, and naïve) immediately after sacrifice. Splenocytes were prepared and stimulated with PPE for 72 h. Cytokines were measured by ELISA. Data are presented as means ± SD for each group of 10 mice. ns: non significant, * p
Figure Legend Snippet: Effect of EPIT on systemic response induced in mice after oral sensitization. (A) Quantity of specific IgE expressed in µg.ml −1 for each group. (B) Quantity of specific IgG2a expressed in µg.ml −1 for each group. (C) Determination of the IgG1/IgG2a ratio expressed for each group. D44 (week 6) concords with the end of sensitization and from D44 to D99 (weeks 7 to 14) with the immunotherapy. Data are expressed as means ± SD for each group of 10 mice. (D–H) Measurement of Th2 cytokine levels (IL-4, IL-5, IL-10, IL-13) and IFN-γ secretion by splenocytes collected from each group of mice (EPIT, Sham, and naïve) immediately after sacrifice. Splenocytes were prepared and stimulated with PPE for 72 h. Cytokines were measured by ELISA. Data are presented as means ± SD for each group of 10 mice. ns: non significant, * p

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

Systemic response induced in mice after oral sensitization analyzed in plasma and spleens. (A) Quantity of specific IgE and IgG2a expressed in µg.ml −1 for each group. Data are expressed as means ± SD for each group mice, D44 after oral sensitization, D89 after the 8-week resting period of peanut free diet. (B–E) Measurement of Th2 cytokine levels (IL-4, IL-5, IL-13) and IFN-γ secretion by splenocytes collected from each group of mice (EPIT, Sham, and naïve) immediately after sacrifice. Splenocytes were prepared and stimulated with PPE for 72 h. Cytokines were measured by ELISA. Data are presented as means ± SD for each group of mice. N: naïve mice, S: sensitized mice. ** p
Figure Legend Snippet: Systemic response induced in mice after oral sensitization analyzed in plasma and spleens. (A) Quantity of specific IgE and IgG2a expressed in µg.ml −1 for each group. Data are expressed as means ± SD for each group mice, D44 after oral sensitization, D89 after the 8-week resting period of peanut free diet. (B–E) Measurement of Th2 cytokine levels (IL-4, IL-5, IL-13) and IFN-γ secretion by splenocytes collected from each group of mice (EPIT, Sham, and naïve) immediately after sacrifice. Splenocytes were prepared and stimulated with PPE for 72 h. Cytokines were measured by ELISA. Data are presented as means ± SD for each group of mice. N: naïve mice, S: sensitized mice. ** p

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

9) Product Images from "Intradermal Delivery of Antigens Enhances Specific IgG and Diminishes IgE Production: Potential Use for Vaccination and Allergy Immunotherapy"

Article Title: Intradermal Delivery of Antigens Enhances Specific IgG and Diminishes IgE Production: Potential Use for Vaccination and Allergy Immunotherapy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0167952

Intradermal antigen injection preferentially diminishes IgE antibody formation. BALB/c mice continuously received 6 times by intradermal (ID) or subcutaneous (SC) injection with 2 μg of OVA in 2 μl saline, or by patch and topical application (epicutaneous) with 200 μg of OVA in 200 μl saline. Blood samples were collected at 3 days before the first sensitization (day-3) and 18 days after the first sensitization (day 18). (A) Timeline for sensitization and blood sampling. (B–D) Concentrations of OVA-specific and total serum IgE (B), IgG1 (C) and IgG2a (D) were determined by ELISA. Each circle represents the concentration of individual 10 mice, and bar shows the mean ± SD. * P
Figure Legend Snippet: Intradermal antigen injection preferentially diminishes IgE antibody formation. BALB/c mice continuously received 6 times by intradermal (ID) or subcutaneous (SC) injection with 2 μg of OVA in 2 μl saline, or by patch and topical application (epicutaneous) with 200 μg of OVA in 200 μl saline. Blood samples were collected at 3 days before the first sensitization (day-3) and 18 days after the first sensitization (day 18). (A) Timeline for sensitization and blood sampling. (B–D) Concentrations of OVA-specific and total serum IgE (B), IgG1 (C) and IgG2a (D) were determined by ELISA. Each circle represents the concentration of individual 10 mice, and bar shows the mean ± SD. * P

Techniques Used: Injection, Mouse Assay, Sampling, Enzyme-linked Immunosorbent Assay, Concentration Assay

Repetitive desensitization via the intradermal route inhibits antigen-specific IgE elevation and enhances antigen-specific IgG production for the long-term. (A) Timeline for sensitization, desensitization and blood sampling. BALB/c mice were used in this experiment. (B–D) Serum concentrations of OVA-specific IgE (B), IgG1 (C) and IgG2a (D) were determined by ELISA. Data are the mean ± SD of 15 mice per group.
Figure Legend Snippet: Repetitive desensitization via the intradermal route inhibits antigen-specific IgE elevation and enhances antigen-specific IgG production for the long-term. (A) Timeline for sensitization, desensitization and blood sampling. BALB/c mice were used in this experiment. (B–D) Serum concentrations of OVA-specific IgE (B), IgG1 (C) and IgG2a (D) were determined by ELISA. Data are the mean ± SD of 15 mice per group.

Techniques Used: Sampling, Mouse Assay, Enzyme-linked Immunosorbent Assay

Langerin positive cells are required for diminishment of IgE production. B6/J and Langerin-DTR mice continuously received 3 times of DT and OVA by intradermal (ID) or subcutaneous (SC) injection with 2 μg of OVA in 2 μl saline. Blood samples were collected at 3 days before the first sensitization (day -3) and 18 days after the first sensitization (day 18). (A) Timeline for injection and blood sampling. (B–D) Serum concentrations of OVA-specific IgE (E), IgG1 (F) and IgG2a (G) were determined by ELISA. Each bar shows the mean ± SD of 8 mice per group. * P
Figure Legend Snippet: Langerin positive cells are required for diminishment of IgE production. B6/J and Langerin-DTR mice continuously received 3 times of DT and OVA by intradermal (ID) or subcutaneous (SC) injection with 2 μg of OVA in 2 μl saline. Blood samples were collected at 3 days before the first sensitization (day -3) and 18 days after the first sensitization (day 18). (A) Timeline for injection and blood sampling. (B–D) Serum concentrations of OVA-specific IgE (E), IgG1 (F) and IgG2a (G) were determined by ELISA. Each bar shows the mean ± SD of 8 mice per group. * P

Techniques Used: Mouse Assay, Injection, Sampling, Enzyme-linked Immunosorbent Assay

Intradermal injection efficiently induces antigen-specific IgG production. BALB/c mice continuously received 6 times by intradermal (ID) or subcutaneous (SC) injection with three different doses, either 0.2, 2 or 20 μg of OVA in 2 μl saline. Blood samples were collected at 3 days before the first sensitization (day-3) and 18 days after the first sensitization (day 18). (A) Timeline for injection and blood sampling. (B–D) Concentrations of OVA-specific serum IgE (B), IgG1 (C) and IgG2a (D) were determined by ELISA. Each circle represents the concentration of individual 7 mice, and bar shows the mean ± SD. * P
Figure Legend Snippet: Intradermal injection efficiently induces antigen-specific IgG production. BALB/c mice continuously received 6 times by intradermal (ID) or subcutaneous (SC) injection with three different doses, either 0.2, 2 or 20 μg of OVA in 2 μl saline. Blood samples were collected at 3 days before the first sensitization (day-3) and 18 days after the first sensitization (day 18). (A) Timeline for injection and blood sampling. (B–D) Concentrations of OVA-specific serum IgE (B), IgG1 (C) and IgG2a (D) were determined by ELISA. Each circle represents the concentration of individual 7 mice, and bar shows the mean ± SD. * P

Techniques Used: Injection, Mouse Assay, Sampling, Enzyme-linked Immunosorbent Assay, Concentration Assay

10) Product Images from "Collagen epitope expression on B cells is sufficient to confer tolerance to collagen-induced arthritis"

Article Title: Collagen epitope expression on B cells is sufficient to confer tolerance to collagen-induced arthritis

Journal: Arthritis Research & Therapy

doi: 10.1186/s13075-016-1037-7

Anti-CII IgG and IgM in serum from LNT-Igk-Ctrl and LNT-Igk-CII mice. Serum levels of a anti-CII IgG, b anti-CII IgG1, c anti-CII IgG2a, d anti-CII IgG2b and e anti-CII IgM in LNT-Igk-Ctrl and LNT-Igk-CII mice measured by ELISA at day 20, day 40 ( n = 10 per group) and day 55 ( n = 6 per group) after immunization. f Change in serum levels of the IgG antibodies to the specific CII epitopes (C1, J1, D3, U1, E10 and F4) measured as Δabsorbance (absorbance at indicated day – mean value at day 0). Serum was obtained from LNT-Igk-Ctrl and LNT-Igk-CII mice at day 0 ( n = 3 per group), day 15 ( n = 6 per group), day 40 ( n = 10 per group) and day 55 ( n = 7 and 6, respectively). Data shown as mean ± SEM. Indicated P values were determined using a two-tailed t test with a Bonferroni correction for multiple comparisons when comparisons were made for multiple anti-recombinant CII-peptide assays. * P
Figure Legend Snippet: Anti-CII IgG and IgM in serum from LNT-Igk-Ctrl and LNT-Igk-CII mice. Serum levels of a anti-CII IgG, b anti-CII IgG1, c anti-CII IgG2a, d anti-CII IgG2b and e anti-CII IgM in LNT-Igk-Ctrl and LNT-Igk-CII mice measured by ELISA at day 20, day 40 ( n = 10 per group) and day 55 ( n = 6 per group) after immunization. f Change in serum levels of the IgG antibodies to the specific CII epitopes (C1, J1, D3, U1, E10 and F4) measured as Δabsorbance (absorbance at indicated day – mean value at day 0). Serum was obtained from LNT-Igk-Ctrl and LNT-Igk-CII mice at day 0 ( n = 3 per group), day 15 ( n = 6 per group), day 40 ( n = 10 per group) and day 55 ( n = 7 and 6, respectively). Data shown as mean ± SEM. Indicated P values were determined using a two-tailed t test with a Bonferroni correction for multiple comparisons when comparisons were made for multiple anti-recombinant CII-peptide assays. * P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Recombinant

11) Product Images from "Antibodies to the Iron Uptake ABC Transporter Lipoproteins PiaA and PiuA Promote Opsonophagocytosis of Streptococcus pneumoniae"

Article Title: Antibodies to the Iron Uptake ABC Transporter Lipoproteins PiaA and PiuA Promote Opsonophagocytosis of Streptococcus pneumoniae

Journal: Infection and Immunity

doi: 10.1128/IAI.73.10.6852-6859.2005

(A and B) Antibody titers to PiuA (A) and PiaA (B) in mouse sera after i.p. vaccination with 10 μg (diagonally hatched bars) or 20 μg (vertically hatched bars) of single antigen or with 5 μg plus 5 μg (horizontally hatched bars) or 10 μg plus 10 μg (open bars) of PiuA and PiaA in combination. (C and D) Serum IgG1 (C) and IgG2a (D) subclass antibody titers to PiuA and PiaA on day 7 (open bars, 7 days after the initial i.p. vaccination), 14 (diagonally hatched bars, 7 days after first booster vaccination), or 21 (vertically hatched bars, 7 days after the second booster i.p. vaccination). Solid bars represent results for sera from mice vaccinated with alum alone, and error bars represent standard deviations. Titers are presented as log 10 values of the reciprocal dilutions giving an OD 405 equal to or greater than 0.30.
Figure Legend Snippet: (A and B) Antibody titers to PiuA (A) and PiaA (B) in mouse sera after i.p. vaccination with 10 μg (diagonally hatched bars) or 20 μg (vertically hatched bars) of single antigen or with 5 μg plus 5 μg (horizontally hatched bars) or 10 μg plus 10 μg (open bars) of PiuA and PiaA in combination. (C and D) Serum IgG1 (C) and IgG2a (D) subclass antibody titers to PiuA and PiaA on day 7 (open bars, 7 days after the initial i.p. vaccination), 14 (diagonally hatched bars, 7 days after first booster vaccination), or 21 (vertically hatched bars, 7 days after the second booster i.p. vaccination). Solid bars represent results for sera from mice vaccinated with alum alone, and error bars represent standard deviations. Titers are presented as log 10 values of the reciprocal dilutions giving an OD 405 equal to or greater than 0.30.

Techniques Used: Mouse Assay

Anti-PiuA and anti-PiaA binding to S. pneumoniae D39 assessed by identifying bacteria coated with antibody by using phycoerythrin-conjugated goat anti-rabbit IgG and flow cytometry. (A) Proportion of wild-type bacteria positive for IgG after incubation in rabbit polyclonal anti-PiuA and anti-PiaA. Solid bars, PBS negative control; open bars, 1/20 dilution of antisera; diagonally hatched bars, 1/5 dilution of antisera; vertically hatched bars, undiluted antisera. For all antiserum dilutions versus PBS, P
Figure Legend Snippet: Anti-PiuA and anti-PiaA binding to S. pneumoniae D39 assessed by identifying bacteria coated with antibody by using phycoerythrin-conjugated goat anti-rabbit IgG and flow cytometry. (A) Proportion of wild-type bacteria positive for IgG after incubation in rabbit polyclonal anti-PiuA and anti-PiaA. Solid bars, PBS negative control; open bars, 1/20 dilution of antisera; diagonally hatched bars, 1/5 dilution of antisera; vertically hatched bars, undiluted antisera. For all antiserum dilutions versus PBS, P

Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Incubation, Negative Control

12) Product Images from "CD28-B7 blockade prevents the development of experimental autoimmune glomerulonephritis"

Article Title: CD28-B7 blockade prevents the development of experimental autoimmune glomerulonephritis

Journal: Journal of Clinical Investigation

doi:

Effect of native CTLA4-Ig or Y100F-Ig treatment on levels of circulating ( a ) IgG ( b ) IgG1, and ( c ) IgG2a anti-GBM antibodies in groups of WKY rats ( n = 5–8) with EAG. Results shown represent the mean of each group at week 4 after immunization: positive control (filled circles), CTLA4-Ig (filled squares), Y100F-Ig (open squares), and negative control (open circles). (* P
Figure Legend Snippet: Effect of native CTLA4-Ig or Y100F-Ig treatment on levels of circulating ( a ) IgG ( b ) IgG1, and ( c ) IgG2a anti-GBM antibodies in groups of WKY rats ( n = 5–8) with EAG. Results shown represent the mean of each group at week 4 after immunization: positive control (filled circles), CTLA4-Ig (filled squares), Y100F-Ig (open squares), and negative control (open circles). (* P

Techniques Used: Positive Control, Negative Control

Direct immunofluorescence of kidney tissue at 4 weeks in WKY rats with EAG. ( a ) Strong linear deposition of IgG along the GBM in an animal given control fusion protein. ( b ) Marked reduction is seen in the deposition of IgG on the GBM in an animal treated with native CTLA4-Ig.
Figure Legend Snippet: Direct immunofluorescence of kidney tissue at 4 weeks in WKY rats with EAG. ( a ) Strong linear deposition of IgG along the GBM in an animal given control fusion protein. ( b ) Marked reduction is seen in the deposition of IgG on the GBM in an animal treated with native CTLA4-Ig.

Techniques Used: Immunofluorescence

13) Product Images from "Antibody-mediated protection against MERS-CoV in the murine model"

Article Title: Antibody-mediated protection against MERS-CoV in the murine model

Journal: Vaccine

doi: 10.1016/j.vaccine.2019.05.074

A. Expression of CD26 was induced in lung tissue by the administration of Ad5hDPP4 (2.5 × 10 8 pfu) to mice by the i.n. route at T 0 . Subsequently, mice were culled in pairs on the days shown and their lungs assayed for the expression of CD26. The plot shows the time-course of CD26 expression from 3 to 17 days post-induction. All data points were normalised for background values from control mice. 6B: Content of MERS-CoV (EMC2012 strain) in murine lungs (pfu/g tissue) determined by RT-PCR at day 3 post-infection, (equivalent to day 4 after passive transfer with murine antisera to RBD-Fc which had previously been shown to neutralise the EMC2012 strain in vitro ). Mice received either a MERS-CoV-specific human IgG (150 μg) or non-specific human IgG (200 μg) in 100 μl /mouse i.p.; or murine antisera to RBD-FC, which had been pooled from 4 murine donors and which was delivered at 1:10 dilution (100 μl/mouse i.p.). Negative control mice received PBS in place of Ad5hDPP4 or antiserum All mice were challenged with MERS-CoV EMC2012 i.n. at 10 4 pfu/mouse. Statistical significance was determined at the p
Figure Legend Snippet: A. Expression of CD26 was induced in lung tissue by the administration of Ad5hDPP4 (2.5 × 10 8 pfu) to mice by the i.n. route at T 0 . Subsequently, mice were culled in pairs on the days shown and their lungs assayed for the expression of CD26. The plot shows the time-course of CD26 expression from 3 to 17 days post-induction. All data points were normalised for background values from control mice. 6B: Content of MERS-CoV (EMC2012 strain) in murine lungs (pfu/g tissue) determined by RT-PCR at day 3 post-infection, (equivalent to day 4 after passive transfer with murine antisera to RBD-Fc which had previously been shown to neutralise the EMC2012 strain in vitro ). Mice received either a MERS-CoV-specific human IgG (150 μg) or non-specific human IgG (200 μg) in 100 μl /mouse i.p.; or murine antisera to RBD-FC, which had been pooled from 4 murine donors and which was delivered at 1:10 dilution (100 μl/mouse i.p.). Negative control mice received PBS in place of Ad5hDPP4 or antiserum All mice were challenged with MERS-CoV EMC2012 i.n. at 10 4 pfu/mouse. Statistical significance was determined at the p

Techniques Used: Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Infection, In Vitro, Negative Control

(A) Serum IgG to RBD-Fc after dual- or single-route immunisation. Mice were immunised with RBD-Fc on PCMC or with RBD-Fc in MF59 s.c. and boosted p.o. with RBD-Fc in the oral formulation, or with RBD-Fc in MF59 s.c., each on day 21. The serum IgG response at days 14 and 35 of the schedule is shown in response to the priming and booster doses. (B) shows the distribution of IgG1 or IgG2a isotypes induced by day 49 of the immunisation schedule. Statistical significance was determined at the p
Figure Legend Snippet: (A) Serum IgG to RBD-Fc after dual- or single-route immunisation. Mice were immunised with RBD-Fc on PCMC or with RBD-Fc in MF59 s.c. and boosted p.o. with RBD-Fc in the oral formulation, or with RBD-Fc in MF59 s.c., each on day 21. The serum IgG response at days 14 and 35 of the schedule is shown in response to the priming and booster doses. (B) shows the distribution of IgG1 or IgG2a isotypes induced by day 49 of the immunisation schedule. Statistical significance was determined at the p

Techniques Used: Mouse Assay

A. Development of RBD-specific murine IgG titres with time in response to RBD-Fc in MF59 or alhydrogel immunisation by the s.c. route on days 0, 10 and 31. The coloured replicates indicate the serum samples from each group assayed (132-blue and 150-purple in the alhydrogel group) and 136-red and 169-green in the MF59 group) and demonstrated to have neutralising activity in vitro for clinical strains of MERS-CoV. (B) shows the in vitro neutralisation of the London1-2012 strain by individual murine antisera to RBD-Fc whilst (C) shows neutralisation of the EMC2012 strain.
Figure Legend Snippet: A. Development of RBD-specific murine IgG titres with time in response to RBD-Fc in MF59 or alhydrogel immunisation by the s.c. route on days 0, 10 and 31. The coloured replicates indicate the serum samples from each group assayed (132-blue and 150-purple in the alhydrogel group) and 136-red and 169-green in the MF59 group) and demonstrated to have neutralising activity in vitro for clinical strains of MERS-CoV. (B) shows the in vitro neutralisation of the London1-2012 strain by individual murine antisera to RBD-Fc whilst (C) shows neutralisation of the EMC2012 strain.

Techniques Used: Activity Assay, In Vitro

14) Product Images from "CD1d Expression in Paneth Cells and Rat Exocrine Pancreas Revealed by Novel Monoclonal Antibodies Which Differentially Affect NKT Cell Activation"

Article Title: CD1d Expression in Paneth Cells and Rat Exocrine Pancreas Revealed by Novel Monoclonal Antibodies Which Differentially Affect NKT Cell Activation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0013089

Characterization of two novel anti-CD1d monoclonal antibodies. (A) Titration of WTH-1 and WTH-2 mAbs on rat CD1d or mouse CD1d1 Raji transductants (left) and on C57BL/6 or LEW thymocytes (right). Cells were stained with the indicated concentrations of CD1d-specific antibodies (X axis). After washing, antibodies were detected with PE-labeled donkey anti-mouse IgG and analyzed by flow cytometry. On the Y axis, the geometric mean fluorescence intensity (MFI) is shown for the different antibody concentrations. (B) Immunoprecipitation of biotinylated surface proteins with WTH-1 (1), WTH-2 (2) or isotype control antibodies. Immunoprecipitated material was size separated under reducing conditions on a 15% SDS-PAGE, blotted onto a membrane and detected by addition of streptavidin-HRP. (C) Western blot analysis of proteins derived from rat CD1d (r) or mouse CD1d1 (m) transduced cells - left blots - and from rat tissues: spleen (spl), thymus (thy) and pancreas (pan) - right blots -. Proteins were separated on a 10% polyacrylamide gel under non-reducing conditions and blotted to a membrane. CD1d was detected with the WTH-1 or WTH-2 mAbs. The duration of film exposure for the CD1d immunoblots is indicated under the mAb names. After CD1d detection, the blots were stripped and re-probed with a polyclonal ERK2-specific antibody as protein loading control (lower blots).
Figure Legend Snippet: Characterization of two novel anti-CD1d monoclonal antibodies. (A) Titration of WTH-1 and WTH-2 mAbs on rat CD1d or mouse CD1d1 Raji transductants (left) and on C57BL/6 or LEW thymocytes (right). Cells were stained with the indicated concentrations of CD1d-specific antibodies (X axis). After washing, antibodies were detected with PE-labeled donkey anti-mouse IgG and analyzed by flow cytometry. On the Y axis, the geometric mean fluorescence intensity (MFI) is shown for the different antibody concentrations. (B) Immunoprecipitation of biotinylated surface proteins with WTH-1 (1), WTH-2 (2) or isotype control antibodies. Immunoprecipitated material was size separated under reducing conditions on a 15% SDS-PAGE, blotted onto a membrane and detected by addition of streptavidin-HRP. (C) Western blot analysis of proteins derived from rat CD1d (r) or mouse CD1d1 (m) transduced cells - left blots - and from rat tissues: spleen (spl), thymus (thy) and pancreas (pan) - right blots -. Proteins were separated on a 10% polyacrylamide gel under non-reducing conditions and blotted to a membrane. CD1d was detected with the WTH-1 or WTH-2 mAbs. The duration of film exposure for the CD1d immunoblots is indicated under the mAb names. After CD1d detection, the blots were stripped and re-probed with a polyclonal ERK2-specific antibody as protein loading control (lower blots).

Techniques Used: Titration, Staining, Labeling, Flow Cytometry, Cytometry, Fluorescence, Immunoprecipitation, SDS Page, Western Blot, Derivative Assay

CD1d and CD4 expression by MZ B cells, dendritic cells and macrophages from the spleen. One representative of three independent experiments is shown. Dot plots illustrate gating strategies. Numbers indicate the percentages of gated cells. Histograms show CD1d and CD4 expression levels. (A) MZ B cell analysis. In C57BL/6 mice, CD1d was stained with biotinylated WTH-2 mAb + SA-Cy5-PE and CD4 with RM4-APCy. In LEW, CD1d was detected with unconjugated WTH-2 mAb followed by PE-labeled donkey anti-mouse IgG antibody and CD4 with OX-35 labeled with PE-Cy5 unless otherwise indicated. Upper row histograms show gated MZ B cells, whereas, lower row histograms show total lymphocytes. MZ B cells in C57BL/6 mice were identified by gating on CD21 hi (7G6-FITC) and CD23 low/negative (B3B4-PE) cells. In LEW rats, MZ B cells were stained with two different marker combinations: CD45RA (OX-33-FITC)/HIS57-biotin + SA-APCy and IgM (G53-238-FITC)/IgD (MARD-3-biotin + SA-APCy), respectively. Analysis of CD1d and CD4 in MZ B cells defined as HIS57 and CD45RA positive cells was carried out with one single multicolor experiment. Expression of CD1d and CD4 in MZ B cells defined as IgD low and IgM high cells was determined in separated multicolor experiments as both, the CD1d and the CD4 specific antibodies, were visualized with the PE fluorochrome (CD1d: WTH-2 + PE-donkey anti-mouse IgG, CD4: OX-35-PE). (B) CD1d and CD4 expression by LEW dendritic cells (OX-62 + PE donkey anti-mouse IgG secondary antibody) and MZ B cells (HIS57-biotin and SA-APCy). CD1d was detected with WTH-2-FITC and CD4 with OX-35-PE-Cy5. (C) CD1d and CD4 expression by LEW macrophages - defined as CD11b/c + cells (OX-42-PE) - and MZ B cells (HIS57-biotin + SA-APCy). CD1d was stained with unconjugated WTH-2 followed by FITC-labeled donkey anti-mouse IgG. For CD4 detection, OX-35-PE-Cy5 antibody was used.
Figure Legend Snippet: CD1d and CD4 expression by MZ B cells, dendritic cells and macrophages from the spleen. One representative of three independent experiments is shown. Dot plots illustrate gating strategies. Numbers indicate the percentages of gated cells. Histograms show CD1d and CD4 expression levels. (A) MZ B cell analysis. In C57BL/6 mice, CD1d was stained with biotinylated WTH-2 mAb + SA-Cy5-PE and CD4 with RM4-APCy. In LEW, CD1d was detected with unconjugated WTH-2 mAb followed by PE-labeled donkey anti-mouse IgG antibody and CD4 with OX-35 labeled with PE-Cy5 unless otherwise indicated. Upper row histograms show gated MZ B cells, whereas, lower row histograms show total lymphocytes. MZ B cells in C57BL/6 mice were identified by gating on CD21 hi (7G6-FITC) and CD23 low/negative (B3B4-PE) cells. In LEW rats, MZ B cells were stained with two different marker combinations: CD45RA (OX-33-FITC)/HIS57-biotin + SA-APCy and IgM (G53-238-FITC)/IgD (MARD-3-biotin + SA-APCy), respectively. Analysis of CD1d and CD4 in MZ B cells defined as HIS57 and CD45RA positive cells was carried out with one single multicolor experiment. Expression of CD1d and CD4 in MZ B cells defined as IgD low and IgM high cells was determined in separated multicolor experiments as both, the CD1d and the CD4 specific antibodies, were visualized with the PE fluorochrome (CD1d: WTH-2 + PE-donkey anti-mouse IgG, CD4: OX-35-PE). (B) CD1d and CD4 expression by LEW dendritic cells (OX-62 + PE donkey anti-mouse IgG secondary antibody) and MZ B cells (HIS57-biotin and SA-APCy). CD1d was detected with WTH-2-FITC and CD4 with OX-35-PE-Cy5. (C) CD1d and CD4 expression by LEW macrophages - defined as CD11b/c + cells (OX-42-PE) - and MZ B cells (HIS57-biotin + SA-APCy). CD1d was stained with unconjugated WTH-2 followed by FITC-labeled donkey anti-mouse IgG. For CD4 detection, OX-35-PE-Cy5 antibody was used.

Techniques Used: Expressing, Mouse Assay, Staining, Labeling, Marker

Epitope mapping of CD1d-specific mAbs. (A) Alignment of the amino acid sequences of the extracellular domains of the CD1d molecules used in this study. α-helical regions are illustrated in bold. The open triangle points out the localization of the rat CD1d allelism. Mutations in mouse CD1d1 are highlighted with closed triangles. The arrow indicates where mouse/human chimeras were joined. Numbers show rat CD1d amino acid positions. Accession numbers for the amino acid sequences can be found in GenBank: rCD1d ( Rattus norvegicus CD1d), BAA82323; mCD1d1 ( Mus musculus CD1d1), NP_031665; sCD1d1 ( Mus spretus CD1d1), ACM45455; hCD1d1 ( Homo sapiens CD1d), NP_001757. The mCD1d2 ( Mus musculus CD1d2) amino acidic sequence differs in one amino acid in the signal peptide (tryptophan, position −13) from the sequence published under the accession number: P11610. (B) Binding capacity of mAbs to different CD1d molecules. Raji cells were transduced with CD1d molecules using a retroviral expression system. Bicistronic EGFP in the CD1d expression vectors served as reporter gene. Cells were stained with unconjugated WTH-1 or WTH-2 mAbs followed by PE-labeled donkey anti-mouse IgG or PE-labeled 1B1 mAb and were analyzed by flow cytometry. All primary antibodies were used at a final concentration of 250 ng/ml. (C) Localization of relevant amino acids in the CD1d tertiary structure. The model depicts part of the co-crystal of the PBS25 glycolipid and mouse CD1d (PDB: 3GMP) and it was visualized using PDB Swiss Viewer Deep View v4.0 [52] ( http://www.expasy.org/spdbv/ ). The α1 and α2 domains are shown. Gray and green ribbon diagrams highlight the regions constituting the N- and C-terminal parts, respectively, of the chimeric molecules. Spheres visualize the amino acids discussed in the text: aspartic acid (D) 93 in red, methionine (M) 162 in green, valine (V) 118 in gray and arginine (R) 21 in blue.
Figure Legend Snippet: Epitope mapping of CD1d-specific mAbs. (A) Alignment of the amino acid sequences of the extracellular domains of the CD1d molecules used in this study. α-helical regions are illustrated in bold. The open triangle points out the localization of the rat CD1d allelism. Mutations in mouse CD1d1 are highlighted with closed triangles. The arrow indicates where mouse/human chimeras were joined. Numbers show rat CD1d amino acid positions. Accession numbers for the amino acid sequences can be found in GenBank: rCD1d ( Rattus norvegicus CD1d), BAA82323; mCD1d1 ( Mus musculus CD1d1), NP_031665; sCD1d1 ( Mus spretus CD1d1), ACM45455; hCD1d1 ( Homo sapiens CD1d), NP_001757. The mCD1d2 ( Mus musculus CD1d2) amino acidic sequence differs in one amino acid in the signal peptide (tryptophan, position −13) from the sequence published under the accession number: P11610. (B) Binding capacity of mAbs to different CD1d molecules. Raji cells were transduced with CD1d molecules using a retroviral expression system. Bicistronic EGFP in the CD1d expression vectors served as reporter gene. Cells were stained with unconjugated WTH-1 or WTH-2 mAbs followed by PE-labeled donkey anti-mouse IgG or PE-labeled 1B1 mAb and were analyzed by flow cytometry. All primary antibodies were used at a final concentration of 250 ng/ml. (C) Localization of relevant amino acids in the CD1d tertiary structure. The model depicts part of the co-crystal of the PBS25 glycolipid and mouse CD1d (PDB: 3GMP) and it was visualized using PDB Swiss Viewer Deep View v4.0 [52] ( http://www.expasy.org/spdbv/ ). The α1 and α2 domains are shown. Gray and green ribbon diagrams highlight the regions constituting the N- and C-terminal parts, respectively, of the chimeric molecules. Spheres visualize the amino acids discussed in the text: aspartic acid (D) 93 in red, methionine (M) 162 in green, valine (V) 118 in gray and arginine (R) 21 in blue.

Techniques Used: Sequencing, Binding Assay, Transduction, Expressing, Staining, Labeling, Flow Cytometry, Cytometry, Concentration Assay

Comparison of CD1d expression levels by rat and mouse primary cells. Representative data from one out of a total of three experiments are shown. (A) Staining of total thymocytes or splenocytes with biotinylated WTH-2 or isotype control antibodies and SA-PE. Gray and black lines correspond to C57BL/6 and LEW cells, respectively. Filled histograms are control stainings. (B) Co-expression of CD1d and TCR on thymocytes was analyzed by two-color flow cytometry. CD1d was stained using the WTH-2 mAb and PE-labeled donkey anti-mouse IgG. For staining of mouse and rat TCRs, H57-597-APCy and R73-bio + SA-APCy were used respectively. Numbers indicate the MFI of anti-CD1d mAb for the gated populations. (C) CD1d expression by B and T cells in the spleen. In C57BL/6 mice, CD1d was analyzed with the biotinylated WTH-2 mAb followed by SA-PE and in LEW rats, since biotinylated mAbs were used for B and T cell identification, CD1d was stained with unconjugated WTH-2 mAb followed by PE-labeled donkey anti-mouse IgG. Filled histograms are control stainings carried out as WTH-2 stainings but with an isotype control antibody. (Upper row) Relative CD1d expression by B and T cells. Histograms show separate multicolor experiments with same overall CD1d staining intensity ( Fig. S2 ), since both, T and B cells, were identified using APCy visualized antibodies in order to avoid unspecific signal due to fluorescence spectral overlap into the PE channel. The gating strategy and the antibodies used are explained in detail in figure S2 . Gray and black lines correspond to B and T cells, respectively. (Lower row) Histograms show CD1d expression by CD4 and CD8 positive T cells. The gating strategy and used antibodies are explained in detail in the figure S3 . Gray and black lines represent CD4 + and CD8 + T cells, respectively.
Figure Legend Snippet: Comparison of CD1d expression levels by rat and mouse primary cells. Representative data from one out of a total of three experiments are shown. (A) Staining of total thymocytes or splenocytes with biotinylated WTH-2 or isotype control antibodies and SA-PE. Gray and black lines correspond to C57BL/6 and LEW cells, respectively. Filled histograms are control stainings. (B) Co-expression of CD1d and TCR on thymocytes was analyzed by two-color flow cytometry. CD1d was stained using the WTH-2 mAb and PE-labeled donkey anti-mouse IgG. For staining of mouse and rat TCRs, H57-597-APCy and R73-bio + SA-APCy were used respectively. Numbers indicate the MFI of anti-CD1d mAb for the gated populations. (C) CD1d expression by B and T cells in the spleen. In C57BL/6 mice, CD1d was analyzed with the biotinylated WTH-2 mAb followed by SA-PE and in LEW rats, since biotinylated mAbs were used for B and T cell identification, CD1d was stained with unconjugated WTH-2 mAb followed by PE-labeled donkey anti-mouse IgG. Filled histograms are control stainings carried out as WTH-2 stainings but with an isotype control antibody. (Upper row) Relative CD1d expression by B and T cells. Histograms show separate multicolor experiments with same overall CD1d staining intensity ( Fig. S2 ), since both, T and B cells, were identified using APCy visualized antibodies in order to avoid unspecific signal due to fluorescence spectral overlap into the PE channel. The gating strategy and the antibodies used are explained in detail in figure S2 . Gray and black lines correspond to B and T cells, respectively. (Lower row) Histograms show CD1d expression by CD4 and CD8 positive T cells. The gating strategy and used antibodies are explained in detail in the figure S3 . Gray and black lines represent CD4 + and CD8 + T cells, respectively.

Techniques Used: Expressing, Staining, Flow Cytometry, Cytometry, Labeling, Mouse Assay, Fluorescence

15) Product Images from "IL-4-STAT6 Signal Transduction-Dependent Induction of the Clinical Phase of Sjögren’s Syndrome-Like Disease of the Nonobese Diabetic Mouse"

Article Title: IL-4-STAT6 Signal Transduction-Dependent Induction of the Clinical Phase of Sjögren’s Syndrome-Like Disease of the Nonobese Diabetic Mouse

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi:

Detection of M3R isotypic autoantibodies by immunofluorescence. Sera collected from NOD.B10- H2 b .C- Stat6 +/+ and NOD.B10- H2 b .C- Stat6 −/− ) for 1 h in a humidified chamber at room temperature. The cells were washed five times (5 min each wash) with PBS, then incubated 30 min at room temperature with FITC-conjugated secondary Abs specific for IgM ( A and B ), IgG ( C and D ), IgG2b ( E and F ), or IgG1 ( G and H ) isotypes diluted 1/100 (Serotec). Cells were again washed and visualized using a Zeiss Axiovert 200 M microscope at ×100 magnification. Images represent an exposure time of 25 ms.
Figure Legend Snippet: Detection of M3R isotypic autoantibodies by immunofluorescence. Sera collected from NOD.B10- H2 b .C- Stat6 +/+ and NOD.B10- H2 b .C- Stat6 −/− ) for 1 h in a humidified chamber at room temperature. The cells were washed five times (5 min each wash) with PBS, then incubated 30 min at room temperature with FITC-conjugated secondary Abs specific for IgM ( A and B ), IgG ( C and D ), IgG2b ( E and F ), or IgG1 ( G and H ) isotypes diluted 1/100 (Serotec). Cells were again washed and visualized using a Zeiss Axiovert 200 M microscope at ×100 magnification. Images represent an exposure time of 25 ms.

Techniques Used: Immunofluorescence, Incubation, Microscopy, Mass Spectrometry

Salivary flow rates of C57BL/6 mice after injections of IgG isolated from NOD.B10- H2 b .C- Stat6 +/+ or NOD.B10- H2 b .C- Stat6 −/− mice. Temporal changes in salivary flow rates of male C57BL/6 mice injected with whole IgG collected from individual 6-wk-old NOD.B10- H2 b .C- Stat6 +/+ ( n = 4F) or NOD.B10- H2 b .C- Stat6 −/− ( n = 5M and 3F) mice ( A ) and 16-wk-old NOD.B10- H2 b .C- Stat6 +/+ ( n = 5F) and NOD.B10- H2 b .C- Stat6 −/− ( n = 2M and 3F) mice ( B ). Mice were injected i.p. with 500 µg of fractionated IgG. All values are expressed as mean salivary flow rates ± SEM. Statistical analysis was performed by two-tailed, paired Student t test using GraphPad InStat software (*, p
Figure Legend Snippet: Salivary flow rates of C57BL/6 mice after injections of IgG isolated from NOD.B10- H2 b .C- Stat6 +/+ or NOD.B10- H2 b .C- Stat6 −/− mice. Temporal changes in salivary flow rates of male C57BL/6 mice injected with whole IgG collected from individual 6-wk-old NOD.B10- H2 b .C- Stat6 +/+ ( n = 4F) or NOD.B10- H2 b .C- Stat6 −/− ( n = 5M and 3F) mice ( A ) and 16-wk-old NOD.B10- H2 b .C- Stat6 +/+ ( n = 5F) and NOD.B10- H2 b .C- Stat6 −/− ( n = 2M and 3F) mice ( B ). Mice were injected i.p. with 500 µg of fractionated IgG. All values are expressed as mean salivary flow rates ± SEM. Statistical analysis was performed by two-tailed, paired Student t test using GraphPad InStat software (*, p

Techniques Used: Flow Cytometry, Mouse Assay, Isolation, Injection, Two Tailed Test, Software

16) Product Images from "Effects of Different Adjuvants in the Context of Intramuscular and Intranasal Routes on Humoral and Cellular Immune Responses Induced by Detergent-Split A/H3N2 Influenza Vaccines in Mice"

Article Title: Effects of Different Adjuvants in the Context of Intramuscular and Intranasal Routes on Humoral and Cellular Immune Responses Induced by Detergent-Split A/H3N2 Influenza Vaccines in Mice

Journal: Clinical and Vaccine Immunology : CVI

doi: 10.1128/CVI.05441-11

Serum IgG1 (a) and IgG2a (b) antibody isotype responses and IgG1/IgG2a ratios (c) in groups of 8 mice immunized intramuscularly (i.m.) or intranasally (i.n.) with nonadjuvanted and adjuvanted A/New York/55/2004 detergent-split vaccines. Titers against
Figure Legend Snippet: Serum IgG1 (a) and IgG2a (b) antibody isotype responses and IgG1/IgG2a ratios (c) in groups of 8 mice immunized intramuscularly (i.m.) or intranasally (i.n.) with nonadjuvanted and adjuvanted A/New York/55/2004 detergent-split vaccines. Titers against

Techniques Used: Mouse Assay

17) Product Images from "Molecular Signatures of the Evolving Immune Response in Mice following a Bordetella pertussis Infection"

Article Title: Molecular Signatures of the Evolving Immune Response in Mice following a Bordetella pertussis Infection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0104548

Antibody profiling after B. pertussis infection. Antibody responses were determined in mouse sera after intranasal infection by whole cell B. pertussis ELISA or MIA and expressed as OD450 or Fluorescent Intensity (F.I.) respectively. (A) IgM against OMV B1917 were absent. (B) Whole cell B. pertussis ELISA indicated IgG antibody formation 10–14 days p.i., which increased until day 28 days p.i. (C–G) Levels of total IgG and individual subtypes (IgG1, IgG2a, IgG2b and IgG3) against OMV B1917 indicated presence of multiple subclasses. (H) Anti-OMV IgA antibodies in serum were induced after 14–28 days p.i. (I) In lung lysates, anti-OMV IgA antibodies were detected 14 and 28 days p.i. I p -values were determined by one-way ANOVA with multiple comparison compared to naive mice (* = p
Figure Legend Snippet: Antibody profiling after B. pertussis infection. Antibody responses were determined in mouse sera after intranasal infection by whole cell B. pertussis ELISA or MIA and expressed as OD450 or Fluorescent Intensity (F.I.) respectively. (A) IgM against OMV B1917 were absent. (B) Whole cell B. pertussis ELISA indicated IgG antibody formation 10–14 days p.i., which increased until day 28 days p.i. (C–G) Levels of total IgG and individual subtypes (IgG1, IgG2a, IgG2b and IgG3) against OMV B1917 indicated presence of multiple subclasses. (H) Anti-OMV IgA antibodies in serum were induced after 14–28 days p.i. (I) In lung lysates, anti-OMV IgA antibodies were detected 14 and 28 days p.i. I p -values were determined by one-way ANOVA with multiple comparison compared to naive mice (* = p

Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, Mouse Assay

18) Product Images from "The Transmembrane Form of the CX3CL1 Chemokine Fractalkine Is Expressed Predominantly by Epithelial Cells in Vivo"

Article Title: The Transmembrane Form of the CX3CL1 Chemokine Fractalkine Is Expressed Predominantly by Epithelial Cells in Vivo

Journal: The American Journal of Pathology

doi:

Distinguishing between cleaved and membrane-tethered fractalkine, generation of specific reagents. A: along with samples of supernatant taken from fractalkine-transfected CHO-K1 cells, were run on 7.5% acrylamide gels under standard reducing conditions. Samples were transferred to nitrocellulose membranes and identical membranes probed using goat anti-fractalkine polyclonal reagent (goat α-Fkn, R D Systems) ( lanes 1–3 ) or chicken anti-C-peptide polyclonal reagent (chicken α-C-pep, lanes 4–6 ). The goat α-Fkn reagent is reactive against the chemokine domain of the molecule and specifically detects twobands at the predicted size of 95 kd ( lane 2 , asterisk ). These two bands are also detected by the chicken α-C-pep reagent ( lane 5 , asterisk ). In addition, these reagents discriminate between cleaved and intact forms of the molecule as the goat α-Fkn detects the cleaved form of fractalkine within transfected cell supernatant ( lane 3 , 85 to 90 kd), whereas the chicken α-C-pep does not ( lane 6 ). Furthermore, the goat α-Fkn detects one larger ( lane 2 , 100 kd) and two smaller bands ( lane 2 , 75 and 66 kd) within transfected CHO-K1 samples that are not detected by the chicken α-C-pep ( lane 5 ). The larger band may be nonspecific because it has no counterpart detected by the chicken α-C-pep. The two smaller bands may indicate partially degraded forms of fractalkine, still containing the N-terminus chemokine domain. B–I: The specificity of a range of anti-fractalkine antibodies was evaluated by immunohistochemistry. Cytospins were prepared from NIH/3T3 cells transiently transfected as above, with fractalkine (3T3-Fkn) and were stained as follows. B: 3T3-Fkn stained with mouse IgG 1 control as a control for C . C: 3T3-Fkn stained with mouse anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; R D Systems) mAb. D: 3T3-Fkn stained with no primary antibody as a control for E and F . E: 3T3-Fkn stained with goat α-Fkn. F: 3T3-Fkn stained with chicken α-C-pep. G: 3T3-Fkn stained with rabbit IgG as a control for H and I . H: 3T3-Fkn stained with rabbit α-C-peptide. I: Note that although there is light nonspecific staining of the nucleus within the control sections ( B , D , and F ) this is in marked contrast to the strong cell surface staining in sections stained with the specific reagents. Similar results were obtained using transfected CHO-K1 cells and via immunofluorescence. Original magnification, ×400.
Figure Legend Snippet: Distinguishing between cleaved and membrane-tethered fractalkine, generation of specific reagents. A: along with samples of supernatant taken from fractalkine-transfected CHO-K1 cells, were run on 7.5% acrylamide gels under standard reducing conditions. Samples were transferred to nitrocellulose membranes and identical membranes probed using goat anti-fractalkine polyclonal reagent (goat α-Fkn, R D Systems) ( lanes 1–3 ) or chicken anti-C-peptide polyclonal reagent (chicken α-C-pep, lanes 4–6 ). The goat α-Fkn reagent is reactive against the chemokine domain of the molecule and specifically detects twobands at the predicted size of 95 kd ( lane 2 , asterisk ). These two bands are also detected by the chicken α-C-pep reagent ( lane 5 , asterisk ). In addition, these reagents discriminate between cleaved and intact forms of the molecule as the goat α-Fkn detects the cleaved form of fractalkine within transfected cell supernatant ( lane 3 , 85 to 90 kd), whereas the chicken α-C-pep does not ( lane 6 ). Furthermore, the goat α-Fkn detects one larger ( lane 2 , 100 kd) and two smaller bands ( lane 2 , 75 and 66 kd) within transfected CHO-K1 samples that are not detected by the chicken α-C-pep ( lane 5 ). The larger band may be nonspecific because it has no counterpart detected by the chicken α-C-pep. The two smaller bands may indicate partially degraded forms of fractalkine, still containing the N-terminus chemokine domain. B–I: The specificity of a range of anti-fractalkine antibodies was evaluated by immunohistochemistry. Cytospins were prepared from NIH/3T3 cells transiently transfected as above, with fractalkine (3T3-Fkn) and were stained as follows. B: 3T3-Fkn stained with mouse IgG 1 control as a control for C . C: 3T3-Fkn stained with mouse anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; R D Systems) mAb. D: 3T3-Fkn stained with no primary antibody as a control for E and F . E: 3T3-Fkn stained with goat α-Fkn. F: 3T3-Fkn stained with chicken α-C-pep. G: 3T3-Fkn stained with rabbit IgG as a control for H and I . H: 3T3-Fkn stained with rabbit α-C-peptide. I: Note that although there is light nonspecific staining of the nucleus within the control sections ( B , D , and F ) this is in marked contrast to the strong cell surface staining in sections stained with the specific reagents. Similar results were obtained using transfected CHO-K1 cells and via immunofluorescence. Original magnification, ×400.

Techniques Used: Transfection, Immunohistochemistry, Staining, Immunofluorescence

The transmembrane form of fractalkine is expressed by the human colorectal adenocarcinoma cell line, DLD-1. A: DLD-1, cells were grown to confluence on glass coverslips and stained using indirect immunofluorescence for transmembrane-expressed fractalkine using the anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; green) mAb and rabbit anti-C-peptide reagent (α-C-pep; red). Strong double labeling (orange) occurred on a subset of cells where the intracellular epitope was most strongly expressed. Lower levels of anti-chemokine domain staining could be detected on most cells. B: Anti-chemokine domain reagent specificity was demonstrated by double labeling using an isotype control antibody for the anti-chemokine mAb (green) and α-C-pep (red). α-C-pep staining was also competed out by addition of 10× molar excess of the immunizing peptide (data not shown). C: The α-Fkn (green) but not α-C-pep staining (red) couldbe competed totally by pre-incubation with a 10× molar excess of recombinant human fractalkine chemokine domain (rhFkn; 362-CX-025; R D Systems). D: Cells were double-labeled with α-cytokeratin (clone AE1/AE3, DAKO; green). Original magnifications, ×400 ( A–D ). E: Total RNA was prepared from DLD-1 and HUVECs cultured with or without 10 U/ml TNF-α. RNA was reverse-transcribed and triplicate 25 ng cDNA samples subjected to PCR reactions using primers specific for fractalkine (Fkn) or HPRT. There was no fractalkine or HPRT signal amplified in reverse transcriptase samples (data not shown). F: DLD-1 cells were permeabilized and stained using i) mouse α-Fkn (clone 51636.11) mAb or control mouse IgG 1 or rabbit IgG, and fractalkine expression analyzed by FACS. The bold trace shows the fluorescence of cells stained with the specific antibody, whereas the normal trace shows the background fluorescence of cells stained with the control reagent.
Figure Legend Snippet: The transmembrane form of fractalkine is expressed by the human colorectal adenocarcinoma cell line, DLD-1. A: DLD-1, cells were grown to confluence on glass coverslips and stained using indirect immunofluorescence for transmembrane-expressed fractalkine using the anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; green) mAb and rabbit anti-C-peptide reagent (α-C-pep; red). Strong double labeling (orange) occurred on a subset of cells where the intracellular epitope was most strongly expressed. Lower levels of anti-chemokine domain staining could be detected on most cells. B: Anti-chemokine domain reagent specificity was demonstrated by double labeling using an isotype control antibody for the anti-chemokine mAb (green) and α-C-pep (red). α-C-pep staining was also competed out by addition of 10× molar excess of the immunizing peptide (data not shown). C: The α-Fkn (green) but not α-C-pep staining (red) couldbe competed totally by pre-incubation with a 10× molar excess of recombinant human fractalkine chemokine domain (rhFkn; 362-CX-025; R D Systems). D: Cells were double-labeled with α-cytokeratin (clone AE1/AE3, DAKO; green). Original magnifications, ×400 ( A–D ). E: Total RNA was prepared from DLD-1 and HUVECs cultured with or without 10 U/ml TNF-α. RNA was reverse-transcribed and triplicate 25 ng cDNA samples subjected to PCR reactions using primers specific for fractalkine (Fkn) or HPRT. There was no fractalkine or HPRT signal amplified in reverse transcriptase samples (data not shown). F: DLD-1 cells were permeabilized and stained using i) mouse α-Fkn (clone 51636.11) mAb or control mouse IgG 1 or rabbit IgG, and fractalkine expression analyzed by FACS. The bold trace shows the fluorescence of cells stained with the specific antibody, whereas the normal trace shows the background fluorescence of cells stained with the control reagent.

Techniques Used: Staining, Immunofluorescence, Labeling, Incubation, Recombinant, Cell Culture, Polymerase Chain Reaction, Amplification, Expressing, FACS, Fluorescence

19) Product Images from "Antibody-mediated protection against MERS-CoV in the murine model "

Article Title: Antibody-mediated protection against MERS-CoV in the murine model

Journal: Vaccine

doi: 10.1016/j.vaccine.2019.05.074

A. Expression of CD26 was induced in lung tissue by the administration of Ad5hDPP4 (2.5 × 10 8 pfu) to mice by the i.n. route at T 0 . Subsequently, mice were culled in pairs on the days shown and their lungs assayed for the expression of CD26. The plot shows the time-course of CD26 expression from 3 to 17 days post-induction. All data points were normalised for background values from control mice. 6B: Content of MERS-CoV (EMC2012 strain) in murine lungs (pfu/g tissue) determined by RT-PCR at day 3 post-infection, (equivalent to day 4 after passive transfer with murine antisera to RBD-Fc which had previously been shown to neutralise the EMC2012 strain in vitro ). Mice received either a MERS-CoV-specific human IgG (150 μg) or non-specific human IgG (200 μg) in 100 μl /mouse i.p.; or murine antisera to RBD-FC, which had been pooled from 4 murine donors and which was delivered at 1:10 dilution (100 μl/mouse i.p.). Negative control mice received PBS in place of Ad5hDPP4 or antiserum All mice were challenged with MERS-CoV EMC2012 i.n. at 10 4 pfu/mouse. Statistical significance was determined at the p
Figure Legend Snippet: A. Expression of CD26 was induced in lung tissue by the administration of Ad5hDPP4 (2.5 × 10 8 pfu) to mice by the i.n. route at T 0 . Subsequently, mice were culled in pairs on the days shown and their lungs assayed for the expression of CD26. The plot shows the time-course of CD26 expression from 3 to 17 days post-induction. All data points were normalised for background values from control mice. 6B: Content of MERS-CoV (EMC2012 strain) in murine lungs (pfu/g tissue) determined by RT-PCR at day 3 post-infection, (equivalent to day 4 after passive transfer with murine antisera to RBD-Fc which had previously been shown to neutralise the EMC2012 strain in vitro ). Mice received either a MERS-CoV-specific human IgG (150 μg) or non-specific human IgG (200 μg) in 100 μl /mouse i.p.; or murine antisera to RBD-FC, which had been pooled from 4 murine donors and which was delivered at 1:10 dilution (100 μl/mouse i.p.). Negative control mice received PBS in place of Ad5hDPP4 or antiserum All mice were challenged with MERS-CoV EMC2012 i.n. at 10 4 pfu/mouse. Statistical significance was determined at the p

Techniques Used: Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Infection, In Vitro, Negative Control

(A) Serum IgG to RBD-Fc after dual- or single-route immunisation. Mice were immunised with RBD-Fc on PCMC or with RBD-Fc in MF59 s.c. and boosted p.o. with RBD-Fc in the oral formulation, or with RBD-Fc in MF59 s.c., each on day 21. The serum IgG response at days 14 and 35 of the schedule is shown in response to the priming and booster doses. (B) shows the distribution of IgG1 or IgG2a isotypes induced by day 49 of the immunisation schedule. Statistical significance was determined at the p
Figure Legend Snippet: (A) Serum IgG to RBD-Fc after dual- or single-route immunisation. Mice were immunised with RBD-Fc on PCMC or with RBD-Fc in MF59 s.c. and boosted p.o. with RBD-Fc in the oral formulation, or with RBD-Fc in MF59 s.c., each on day 21. The serum IgG response at days 14 and 35 of the schedule is shown in response to the priming and booster doses. (B) shows the distribution of IgG1 or IgG2a isotypes induced by day 49 of the immunisation schedule. Statistical significance was determined at the p

Techniques Used: Mouse Assay

A. Development of RBD-specific murine IgG titres with time in response to RBD-Fc in MF59 or alhydrogel immunisation by the s.c. route on days 0, 10 and 31. The coloured replicates indicate the serum samples from each group assayed (132-blue and 150-purple in the alhydrogel group) and 136-red and 169-green in the MF59 group) and demonstrated to have neutralising activity in vitro for clinical strains of MERS-CoV. (B) shows the in vitro neutralisation of the London1-2012 strain by individual murine antisera to RBD-Fc whilst (C) shows neutralisation of the EMC2012 strain.
Figure Legend Snippet: A. Development of RBD-specific murine IgG titres with time in response to RBD-Fc in MF59 or alhydrogel immunisation by the s.c. route on days 0, 10 and 31. The coloured replicates indicate the serum samples from each group assayed (132-blue and 150-purple in the alhydrogel group) and 136-red and 169-green in the MF59 group) and demonstrated to have neutralising activity in vitro for clinical strains of MERS-CoV. (B) shows the in vitro neutralisation of the London1-2012 strain by individual murine antisera to RBD-Fc whilst (C) shows neutralisation of the EMC2012 strain.

Techniques Used: Activity Assay, In Vitro

20) Product Images from "Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice"

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

Journal: Anesthesiology

doi: 10.1097/ALN.0b013e3182a950da

Isoflurane induces interleukin (IL)-11 in human proximal tubule (HK-2) cells via transforming growth factor-beta 1 (TGF-β1) A. IL-11 messenger ribonucleic acid (mRNA) (detected with reverse transcription polymerase chain reaction (RTPCR)) expression in HK-2 cells treated with 2.5% isoflurane for 6 h (N = 6). Representative images (top) and band intensity quantifications (bottom) expressed as fold increases in IL-11 expression over carrier gas plus immunoglobulin G (IgG) isotype antibody treated controls. B. IL-11 protein (detected with enzyme-linked immunosorbent assay (ELISA)) expression in HK-2 cells treated with 2.5% isoflurane for 6 h (N = 6). * P
Figure Legend Snippet: Isoflurane induces interleukin (IL)-11 in human proximal tubule (HK-2) cells via transforming growth factor-beta 1 (TGF-β1) A. IL-11 messenger ribonucleic acid (mRNA) (detected with reverse transcription polymerase chain reaction (RTPCR)) expression in HK-2 cells treated with 2.5% isoflurane for 6 h (N = 6). Representative images (top) and band intensity quantifications (bottom) expressed as fold increases in IL-11 expression over carrier gas plus immunoglobulin G (IgG) isotype antibody treated controls. B. IL-11 protein (detected with enzyme-linked immunosorbent assay (ELISA)) expression in HK-2 cells treated with 2.5% isoflurane for 6 h (N = 6). * P

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

Isoflurane induces interleukin (IL)-11 messenger ribonucleic acid (mRNA) expression in primary culture of mouse proximal tubule cells via transforming growth factor-beta 1 (TGF-β1) IL-11 mRNA (reverse transcription polymerase chain reaction (RTPCR)) expression in primary culture of mouse proximal tubule cells treated with 2.5% isoflurane for 6 h (N = 4). Representative images (top) and band intensity quantifications (bottom) expressed as fold increases in IL-11 expression over carrier gas and immunoglobulin G (IgG) isotype antibody treated controls. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was also quantified to normalize lane loading. * P
Figure Legend Snippet: Isoflurane induces interleukin (IL)-11 messenger ribonucleic acid (mRNA) expression in primary culture of mouse proximal tubule cells via transforming growth factor-beta 1 (TGF-β1) IL-11 mRNA (reverse transcription polymerase chain reaction (RTPCR)) expression in primary culture of mouse proximal tubule cells treated with 2.5% isoflurane for 6 h (N = 4). Representative images (top) and band intensity quantifications (bottom) expressed as fold increases in IL-11 expression over carrier gas and immunoglobulin G (IgG) isotype antibody treated controls. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was also quantified to normalize lane loading. * P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

21) Product Images from "Antibodies to the Iron Uptake ABC Transporter Lipoproteins PiaA and PiuA Promote Opsonophagocytosis of Streptococcus pneumoniae"

Article Title: Antibodies to the Iron Uptake ABC Transporter Lipoproteins PiaA and PiuA Promote Opsonophagocytosis of Streptococcus pneumoniae

Journal: Infection and Immunity

doi: 10.1128/IAI.73.10.6852-6859.2005

(A and B) Antibody titers to PiuA (A) and PiaA (B) in mouse sera after i.p. vaccination with 10 μg (diagonally hatched bars) or 20 μg (vertically hatched bars) of single antigen or with 5 μg plus 5 μg (horizontally hatched bars) or 10 μg plus 10 μg (open bars) of PiuA and PiaA in combination. (C and D) Serum IgG1 (C) and IgG2a (D) subclass antibody titers to PiuA and PiaA on day 7 (open bars, 7 days after the initial i.p. vaccination), 14 (diagonally hatched bars, 7 days after first booster vaccination), or 21 (vertically hatched bars, 7 days after the second booster i.p. vaccination). Solid bars represent results for sera from mice vaccinated with alum alone, and error bars represent standard deviations. Titers are presented as log 10 values of the reciprocal dilutions giving an OD 405 equal to or greater than 0.30.
Figure Legend Snippet: (A and B) Antibody titers to PiuA (A) and PiaA (B) in mouse sera after i.p. vaccination with 10 μg (diagonally hatched bars) or 20 μg (vertically hatched bars) of single antigen or with 5 μg plus 5 μg (horizontally hatched bars) or 10 μg plus 10 μg (open bars) of PiuA and PiaA in combination. (C and D) Serum IgG1 (C) and IgG2a (D) subclass antibody titers to PiuA and PiaA on day 7 (open bars, 7 days after the initial i.p. vaccination), 14 (diagonally hatched bars, 7 days after first booster vaccination), or 21 (vertically hatched bars, 7 days after the second booster i.p. vaccination). Solid bars represent results for sera from mice vaccinated with alum alone, and error bars represent standard deviations. Titers are presented as log 10 values of the reciprocal dilutions giving an OD 405 equal to or greater than 0.30.

Techniques Used: Mouse Assay

Anti-PiuA and anti-PiaA binding to S. pneumoniae D39 assessed by identifying bacteria coated with antibody by using phycoerythrin-conjugated goat anti-rabbit IgG and flow cytometry. (A) Proportion of wild-type bacteria positive for IgG after incubation in rabbit polyclonal anti-PiuA and anti-PiaA. Solid bars, PBS negative control; open bars, 1/20 dilution of antisera; diagonally hatched bars, 1/5 dilution of antisera; vertically hatched bars, undiluted antisera. For all antiserum dilutions versus PBS, P
Figure Legend Snippet: Anti-PiuA and anti-PiaA binding to S. pneumoniae D39 assessed by identifying bacteria coated with antibody by using phycoerythrin-conjugated goat anti-rabbit IgG and flow cytometry. (A) Proportion of wild-type bacteria positive for IgG after incubation in rabbit polyclonal anti-PiuA and anti-PiaA. Solid bars, PBS negative control; open bars, 1/20 dilution of antisera; diagonally hatched bars, 1/5 dilution of antisera; vertically hatched bars, undiluted antisera. For all antiserum dilutions versus PBS, P

Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Incubation, Negative Control

22) Product Images from "Modulation of experimental blood stage malaria through blockade of the B7/CD28 T-cell costimulatory pathway"

Article Title: Modulation of experimental blood stage malaria through blockade of the B7/CD28 T-cell costimulatory pathway

Journal: Immunology

doi: 10.1046/j.1365-2567.1999.00718.x

Courses of infection of P. chabaudi in NIH mice treated with anti-CD80 and/or anti-CD86 mAb (regimen described in the Materials and Methods). Control mice received an irrelevant mAb of identical rat IgG2a isotype, and had infections similar to mice either given rat IgG or left untreated (not shown). Mice were infected with 1×10 5 pRBC on day 0 and parasitaemias were determined daily by examination of Giemsa-stained thin blood smears. Values are medians of parasitaemias of nine mice per group. Interquartile ranges (not shown) were
Figure Legend Snippet: Courses of infection of P. chabaudi in NIH mice treated with anti-CD80 and/or anti-CD86 mAb (regimen described in the Materials and Methods). Control mice received an irrelevant mAb of identical rat IgG2a isotype, and had infections similar to mice either given rat IgG or left untreated (not shown). Mice were infected with 1×10 5 pRBC on day 0 and parasitaemias were determined daily by examination of Giemsa-stained thin blood smears. Values are medians of parasitaemias of nine mice per group. Interquartile ranges (not shown) were

Techniques Used: Infection, Mouse Assay, Staining

Effect of B7/CD28 blockade on the development of malaria-specific IgG in P. chabaudi -infected NIH mice. Serum was collected on the days indicated and measured for P. chabaudi -specific antibody levels using IgG1 (a) and IgG2a (b) isotype-specific ELISA. Data are expressed as the mean reciprocal antibody titre±1 SD ( n =4) for serum samples pooled from 18 mice per time-point, derived from three similar experiments. ND=not detected.
Figure Legend Snippet: Effect of B7/CD28 blockade on the development of malaria-specific IgG in P. chabaudi -infected NIH mice. Serum was collected on the days indicated and measured for P. chabaudi -specific antibody levels using IgG1 (a) and IgG2a (b) isotype-specific ELISA. Data are expressed as the mean reciprocal antibody titre±1 SD ( n =4) for serum samples pooled from 18 mice per time-point, derived from three similar experiments. ND=not detected.

Techniques Used: Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay

23) Product Images from "Prevention of autoimmune disease due to lymphocyte modulation by the B-subunit of Escherichia coli heat-labile enterotoxin"

Article Title: Prevention of autoimmune disease due to lymphocyte modulation by the B-subunit of Escherichia coli heat-labile enterotoxin

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

doi:

Administration of EtxB leads to a reduction in the levels of all IgG isotypes in the anti-CII response and an altered IgG1/IgG2a ratio. Serum samples from mice ( n = 11) injected either with CII alone ( A ) or CII plus EtxB ( B ) were collected 15 days after immunization and assayed for the presence of anti-bovine CII antibodies of each IgG isotype (as indicated). End point titers of each isotype were calculated by linear regression analysis for each animal and the mean values (±SEM) for each group are shown.
Figure Legend Snippet: Administration of EtxB leads to a reduction in the levels of all IgG isotypes in the anti-CII response and an altered IgG1/IgG2a ratio. Serum samples from mice ( n = 11) injected either with CII alone ( A ) or CII plus EtxB ( B ) were collected 15 days after immunization and assayed for the presence of anti-bovine CII antibodies of each IgG isotype (as indicated). End point titers of each isotype were calculated by linear regression analysis for each animal and the mean values (±SEM) for each group are shown.

Techniques Used: Mouse Assay, Injection

Receptor interaction by EtxB does not prevent T cell reactivity to CII but does reduce the anti-CII antibody response. ( A ) Inguinal lymph node cells were isolated 16 days after injection with collagen and were cultured either in the presence (solid symbols) or absence (open symbols) or 50 μg/ml of heat denatured bovine CII. Identical cultures were established from either EtxB-protected (triangles) or unprotected (squares) mice. Proliferation in these cultures was measured on days 2 to 6 by assay of [ 3 H]thymidine incorporation. ( B ) Serum samples from mice ( n = 11) injected either with CII alone or with CII plus EtxB (as indicated) were collected 15 days after immunization and assayed for the presence of anti-bovine CII antibodies. End point titers of total anti-CII IgG were calculated by linear regression analysis for each animal and are shown (□) along with the mean (bar) for the group.
Figure Legend Snippet: Receptor interaction by EtxB does not prevent T cell reactivity to CII but does reduce the anti-CII antibody response. ( A ) Inguinal lymph node cells were isolated 16 days after injection with collagen and were cultured either in the presence (solid symbols) or absence (open symbols) or 50 μg/ml of heat denatured bovine CII. Identical cultures were established from either EtxB-protected (triangles) or unprotected (squares) mice. Proliferation in these cultures was measured on days 2 to 6 by assay of [ 3 H]thymidine incorporation. ( B ) Serum samples from mice ( n = 11) injected either with CII alone or with CII plus EtxB (as indicated) were collected 15 days after immunization and assayed for the presence of anti-bovine CII antibodies. End point titers of total anti-CII IgG were calculated by linear regression analysis for each animal and are shown (□) along with the mean (bar) for the group.

Techniques Used: Isolation, Injection, Cell Culture, Mouse Assay

24) Product Images from "Immunization with recombinant enolase of Sporothrix spp. (rSsEno) confers effective protection against sporotrichosis in mice"

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

Journal: Scientific Reports

doi: 10.1038/s41598-019-53135-z

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

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

25) Product Images from "Involvement of Innate and Adaptive Immunity in a Murine Model of Coronary Arteritis Mimicking Kawasaki Disease"

Article Title: Involvement of Innate and Adaptive Immunity in a Murine Model of Coronary Arteritis Mimicking Kawasaki Disease

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.0901395

Macrophages are found in LCCWE induced coronary arteritis lesions. 21 days after i. p. injection with LCCWE, wt mice were sacrificed and the hearts were frozen in OCT. IHC was performed using anti-F4/80. A) LCCWE injected wt mice (40X and 150X). B) PBS injected control mice (40X). C) Isotype control rat IgG2b (40X). D) Quantification of F4/80 + cells in the LCCWE induced coronary lesion (n= 3 mice/group).
Figure Legend Snippet: Macrophages are found in LCCWE induced coronary arteritis lesions. 21 days after i. p. injection with LCCWE, wt mice were sacrificed and the hearts were frozen in OCT. IHC was performed using anti-F4/80. A) LCCWE injected wt mice (40X and 150X). B) PBS injected control mice (40X). C) Isotype control rat IgG2b (40X). D) Quantification of F4/80 + cells in the LCCWE induced coronary lesion (n= 3 mice/group).

Techniques Used: Injection, Mouse Assay, Immunohistochemistry

CD3+ cells are found in LCCWE induced coronary arteritis lesions. 21 days after i. p. injection with LCCWE, WT mice were sacrificed and the hearts were frozen in OCT. IHC was performed using anti-CD3. A) LCCWE injected wt mice (40X and 150X). B) PBS injected control mice (40X). C) Isotype control rat IgG2a (40X). D) Quantification of CD3 + cells in the LCCWE induced coronary lesion (n= 3 mice/group).
Figure Legend Snippet: CD3+ cells are found in LCCWE induced coronary arteritis lesions. 21 days after i. p. injection with LCCWE, WT mice were sacrificed and the hearts were frozen in OCT. IHC was performed using anti-CD3. A) LCCWE injected wt mice (40X and 150X). B) PBS injected control mice (40X). C) Isotype control rat IgG2a (40X). D) Quantification of CD3 + cells in the LCCWE induced coronary lesion (n= 3 mice/group).

Techniques Used: Injection, Mouse Assay, Immunohistochemistry

Plasmacytoid dendritic cells are found in LCCWE induced coronary arteritis lesions. 21 days after i. p. injection with LCCWE, wt mice were sacrificed and the hearts were frozen in OCT. IHC was performed using anti-PDCA-1. A) LCCWE injected wt mice (40X and 150X). B) PBS injected control mice (40X). C) Isotype control rat IgG2a (40X). D) Quantification of PDCA-1 + cells in the LCCWE induced coronary lesion (n= 3 mice/group).
Figure Legend Snippet: Plasmacytoid dendritic cells are found in LCCWE induced coronary arteritis lesions. 21 days after i. p. injection with LCCWE, wt mice were sacrificed and the hearts were frozen in OCT. IHC was performed using anti-PDCA-1. A) LCCWE injected wt mice (40X and 150X). B) PBS injected control mice (40X). C) Isotype control rat IgG2a (40X). D) Quantification of PDCA-1 + cells in the LCCWE induced coronary lesion (n= 3 mice/group).

Techniques Used: Injection, Mouse Assay, Immunohistochemistry

Activated myeloid dendritic cells are found in LCCWE induced coronary arteritis lesions. 21 days after i. p. injection with LCCWE, wt mice were sacrificed and the hearts were frozen in OCT. IHC was performed using anti-MIDC-8. A) LCCWE injected wt mice (40X and 150X). B) PBS injected control mice (40X). C) Isotype control rat IgG2a (40X). D) Quantification of MIDC8 + cells in the LCCWE induced coronary lesion (n= 3 mice/group).
Figure Legend Snippet: Activated myeloid dendritic cells are found in LCCWE induced coronary arteritis lesions. 21 days after i. p. injection with LCCWE, wt mice were sacrificed and the hearts were frozen in OCT. IHC was performed using anti-MIDC-8. A) LCCWE injected wt mice (40X and 150X). B) PBS injected control mice (40X). C) Isotype control rat IgG2a (40X). D) Quantification of MIDC8 + cells in the LCCWE induced coronary lesion (n= 3 mice/group).

Techniques Used: Injection, Mouse Assay, Immunohistochemistry

26) Product Images from "Disrupted Adenovirus-Based Vaccines Against Small Addictive Molecules Circumvent Anti-Adenovirus Immunity"

Article Title: Disrupted Adenovirus-Based Vaccines Against Small Addictive Molecules Circumvent Anti-Adenovirus Immunity

Journal: Human Gene Therapy

doi: 10.1089/hum.2012.163

Levels of the addictive drug in brain and serum of dAd5-vaccinated mice with and without pre-existing Ad5 immunity. Mice were challenged with 3 H-labeled drug and brain and serum were collected 1 min later, n=4 mice/group, 9 weeks after dAd5GNE or 10 weeks after dAd5AM1 vaccination. (A) Cocaine levels in the brain (ng/g brain) of untreated and dAd5GNE-vaccinated mice naive or with pre-existing Ad5 immunity. (B) Nicotine levels in the brain (ng/g brain) of untreated and dAd5AM1-vaccinated mice, naive or with pre-existing Ad5 immunity. (C) In the same mice as panel (A) , serum cocaine levels (ng/ml), IgG-bound, and free cocaine levels (ng/ml). (D) In the same mice as panel (B) , serum nicotine levels (ng/ml), IgG-bound, and free nicotine levels (ng/ml).
Figure Legend Snippet: Levels of the addictive drug in brain and serum of dAd5-vaccinated mice with and without pre-existing Ad5 immunity. Mice were challenged with 3 H-labeled drug and brain and serum were collected 1 min later, n=4 mice/group, 9 weeks after dAd5GNE or 10 weeks after dAd5AM1 vaccination. (A) Cocaine levels in the brain (ng/g brain) of untreated and dAd5GNE-vaccinated mice naive or with pre-existing Ad5 immunity. (B) Nicotine levels in the brain (ng/g brain) of untreated and dAd5AM1-vaccinated mice, naive or with pre-existing Ad5 immunity. (C) In the same mice as panel (A) , serum cocaine levels (ng/ml), IgG-bound, and free cocaine levels (ng/ml). (D) In the same mice as panel (B) , serum nicotine levels (ng/ml), IgG-bound, and free nicotine levels (ng/ml).

Techniques Used: Mouse Assay, Labeling

Antibody IgG isotype titers evoked by dAd5-based vaccines in naive and anti-Ad5 preimmune mice. Antibody isotypes in dAd5GNE or dAd5AM1 vaccinated naive and Ad5-immune mice at 5 weeks were evaluated by isotype-specific secondary antibodies on ELISA for IgG1, IgG2a, and IgG2b. (A) Anti-cocaine IgG isotype titers in dAd5GNE-vaccinated mice. (B) Anti-nicotine IgG isotype titers in dAd5AM1-vaccinated mice. Antibody titers are mean values±SEM.
Figure Legend Snippet: Antibody IgG isotype titers evoked by dAd5-based vaccines in naive and anti-Ad5 preimmune mice. Antibody isotypes in dAd5GNE or dAd5AM1 vaccinated naive and Ad5-immune mice at 5 weeks were evaluated by isotype-specific secondary antibodies on ELISA for IgG1, IgG2a, and IgG2b. (A) Anti-cocaine IgG isotype titers in dAd5GNE-vaccinated mice. (B) Anti-nicotine IgG isotype titers in dAd5AM1-vaccinated mice. Antibody titers are mean values±SEM.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

27) Product Images from "The regulatory T cells induction by epicutaneous immunotherapy is sustained and mediates long-term protection from eosinophilic disorders in peanut-sensitized mice"

Article Title: The regulatory T cells induction by epicutaneous immunotherapy is sustained and mediates long-term protection from eosinophilic disorders in peanut-sensitized mice

Journal: Clinical and Experimental Allergy

doi: 10.1111/cea.12312

EPIT-induced desensitization was blocked by anti-CD25 antibodies. (a) Quantity of peanut-specific IgE (left panel) and IgG2a for each group after sensitization, before EPIT (d42) and after the treatment period (d104). (b) Measurement of IL-5 and IFN-γ secretion by splenocytes collected from each group of mice immediately after killing. Splenocytes were stimulated with peanut for 72 h. Cytokines were measured using Bioplex. (c) Proportion of Tregs in the spleen of mice from each group. Sham: peanut-sensitized untreated mice; EPIT: peanut-sensitized mice treated by EPIT; EPIT + αCD25: peanut-sensitized mice treated by EPIT and anti-CD25 antibody. Data are shown as means ± SEM of three independent experiments for each group of mice ( n = 8 in each group). * P
Figure Legend Snippet: EPIT-induced desensitization was blocked by anti-CD25 antibodies. (a) Quantity of peanut-specific IgE (left panel) and IgG2a for each group after sensitization, before EPIT (d42) and after the treatment period (d104). (b) Measurement of IL-5 and IFN-γ secretion by splenocytes collected from each group of mice immediately after killing. Splenocytes were stimulated with peanut for 72 h. Cytokines were measured using Bioplex. (c) Proportion of Tregs in the spleen of mice from each group. Sham: peanut-sensitized untreated mice; EPIT: peanut-sensitized mice treated by EPIT; EPIT + αCD25: peanut-sensitized mice treated by EPIT and anti-CD25 antibody. Data are shown as means ± SEM of three independent experiments for each group of mice ( n = 8 in each group). * P

Techniques Used: Mouse Assay

28) Product Images from "Antibody-mediated protection against MERS-CoV in the murine model "

Article Title: Antibody-mediated protection against MERS-CoV in the murine model

Journal: Vaccine

doi: 10.1016/j.vaccine.2019.05.074

A. Expression of CD26 was induced in lung tissue by the administration of Ad5hDPP4 (2.5 × 10 8 pfu) to mice by the i.n. route at T 0 . Subsequently, mice were culled in pairs on the days shown and their lungs assayed for the expression of CD26. The plot shows the time-course of CD26 expression from 3 to 17 days post-induction. All data points were normalised for background values from control mice. 6B: Content of MERS-CoV (EMC2012 strain) in murine lungs (pfu/g tissue) determined by RT-PCR at day 3 post-infection, (equivalent to day 4 after passive transfer with murine antisera to RBD-Fc which had previously been shown to neutralise the EMC2012 strain in vitro ). Mice received either a MERS-CoV-specific human IgG (150 μg) or non-specific human IgG (200 μg) in 100 μl /mouse i.p.; or murine antisera to RBD-FC, which had been pooled from 4 murine donors and which was delivered at 1:10 dilution (100 μl/mouse i.p.). Negative control mice received PBS in place of Ad5hDPP4 or antiserum All mice were challenged with MERS-CoV EMC2012 i.n. at 10 4 pfu/mouse. Statistical significance was determined at the p
Figure Legend Snippet: A. Expression of CD26 was induced in lung tissue by the administration of Ad5hDPP4 (2.5 × 10 8 pfu) to mice by the i.n. route at T 0 . Subsequently, mice were culled in pairs on the days shown and their lungs assayed for the expression of CD26. The plot shows the time-course of CD26 expression from 3 to 17 days post-induction. All data points were normalised for background values from control mice. 6B: Content of MERS-CoV (EMC2012 strain) in murine lungs (pfu/g tissue) determined by RT-PCR at day 3 post-infection, (equivalent to day 4 after passive transfer with murine antisera to RBD-Fc which had previously been shown to neutralise the EMC2012 strain in vitro ). Mice received either a MERS-CoV-specific human IgG (150 μg) or non-specific human IgG (200 μg) in 100 μl /mouse i.p.; or murine antisera to RBD-FC, which had been pooled from 4 murine donors and which was delivered at 1:10 dilution (100 μl/mouse i.p.). Negative control mice received PBS in place of Ad5hDPP4 or antiserum All mice were challenged with MERS-CoV EMC2012 i.n. at 10 4 pfu/mouse. Statistical significance was determined at the p

Techniques Used: Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Infection, In Vitro, Negative Control

(A) Serum IgG to RBD-Fc after dual- or single-route immunisation. Mice were immunised with RBD-Fc on PCMC or with RBD-Fc in MF59 s.c. and boosted p.o. with RBD-Fc in the oral formulation, or with RBD-Fc in MF59 s.c., each on day 21. The serum IgG response at days 14 and 35 of the schedule is shown in response to the priming and booster doses. (B) shows the distribution of IgG1 or IgG2a isotypes induced by day 49 of the immunisation schedule. Statistical significance was determined at the p
Figure Legend Snippet: (A) Serum IgG to RBD-Fc after dual- or single-route immunisation. Mice were immunised with RBD-Fc on PCMC or with RBD-Fc in MF59 s.c. and boosted p.o. with RBD-Fc in the oral formulation, or with RBD-Fc in MF59 s.c., each on day 21. The serum IgG response at days 14 and 35 of the schedule is shown in response to the priming and booster doses. (B) shows the distribution of IgG1 or IgG2a isotypes induced by day 49 of the immunisation schedule. Statistical significance was determined at the p

Techniques Used: Mouse Assay

A. Development of RBD-specific murine IgG titres with time in response to RBD-Fc in MF59 or alhydrogel immunisation by the s.c. route on days 0, 10 and 31. The coloured replicates indicate the serum samples from each group assayed (132-blue and 150-purple in the alhydrogel group) and 136-red and 169-green in the MF59 group) and demonstrated to have neutralising activity in vitro for clinical strains of MERS-CoV. (B) shows the in vitro neutralisation of the London1-2012 strain by individual murine antisera to RBD-Fc whilst (C) shows neutralisation of the EMC2012 strain.
Figure Legend Snippet: A. Development of RBD-specific murine IgG titres with time in response to RBD-Fc in MF59 or alhydrogel immunisation by the s.c. route on days 0, 10 and 31. The coloured replicates indicate the serum samples from each group assayed (132-blue and 150-purple in the alhydrogel group) and 136-red and 169-green in the MF59 group) and demonstrated to have neutralising activity in vitro for clinical strains of MERS-CoV. (B) shows the in vitro neutralisation of the London1-2012 strain by individual murine antisera to RBD-Fc whilst (C) shows neutralisation of the EMC2012 strain.

Techniques Used: Activity Assay, In Vitro

29) Product Images from "DNA immunization with fusion genes encoding different regions of hepatitis C virus E2 fused to the gene for hepatitis B surface antigen elicits immune responses to both HCV and HBV"

Article Title: DNA immunization with fusion genes encoding different regions of hepatitis C virus E2 fused to the gene for hepatitis B surface antigen elicits immune responses to both HCV and HBV

Journal: World Journal of Gastroenterology

doi: 10.3748/wjg.v8.i3.505

IgG subtypes profile of antibodies against HBsAg and HCV E2. Sera collected from mice 6 weeks after boosting were assayed for the IgG1 and IgG2a antibodies against HBsAg and HCV E2. For each group of 5 mice, titers of IgG1 and IgG2a antibodies were determined individually by serial two-fold dilution titration methods using HRP-conjugated rabbit anti-mouse IgG1 and IgG2a (Serotec Co., Oxford, UK) for detection. (as described in Materials and Methods). The arithmetic mean ± standard deviation (SD) ( n = 5) is shown. (A) Anti-HBs, (B) Anti-HCV E2.
Figure Legend Snippet: IgG subtypes profile of antibodies against HBsAg and HCV E2. Sera collected from mice 6 weeks after boosting were assayed for the IgG1 and IgG2a antibodies against HBsAg and HCV E2. For each group of 5 mice, titers of IgG1 and IgG2a antibodies were determined individually by serial two-fold dilution titration methods using HRP-conjugated rabbit anti-mouse IgG1 and IgG2a (Serotec Co., Oxford, UK) for detection. (as described in Materials and Methods). The arithmetic mean ± standard deviation (SD) ( n = 5) is shown. (A) Anti-HBs, (B) Anti-HCV E2.

Techniques Used: Mouse Assay, Titration, Standard Deviation

30) Product Images from "Focal and segmental glomerulosclerosis induced in mice lacking decay-accelerating factor in T cells"

Article Title: Focal and segmental glomerulosclerosis induced in mice lacking decay-accelerating factor in T cells

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI36000

Abnormal anti-sheep IgG responses occur when DAF is absent outside T cells.
Figure Legend Snippet: Abnormal anti-sheep IgG responses occur when DAF is absent outside T cells.

Techniques Used:

31) Product Images from "A 21.6 kDa tegumental protein of Clonorchis sinensis induces a Th1/Th2 mixed immune response in mice"

Article Title: A 21.6 kDa tegumental protein of Clonorchis sinensis induces a Th1/Th2 mixed immune response in mice

Journal: Immunity, Inflammation and Disease

doi: 10.1002/iid3.235

rCsTegu21.6 induced specific antibody production in C57BL/6 mice. rCsTegu21.6 (with or without alum adjuvant (Alum) or Freund's adjuvant (FA) was used to immunize C57BL/6 mice ( n = 5 per group) three times after two‐week intervals. Total IgG (A) and their isotypes (B) were detected in the immunized serum by ELISA. The results are shown as the mean ± standard error (SEM). * P
Figure Legend Snippet: rCsTegu21.6 induced specific antibody production in C57BL/6 mice. rCsTegu21.6 (with or without alum adjuvant (Alum) or Freund's adjuvant (FA) was used to immunize C57BL/6 mice ( n = 5 per group) three times after two‐week intervals. Total IgG (A) and their isotypes (B) were detected in the immunized serum by ELISA. The results are shown as the mean ± standard error (SEM). * P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

32) Product Images from "Carnauba wax nanoparticles enhance strong systemic and mucosal cellular and humoral immune responses to HIV-gp140 antigen"

Article Title: Carnauba wax nanoparticles enhance strong systemic and mucosal cellular and humoral immune responses to HIV-gp140 antigen

Journal: Vaccine

doi: 10.1016/j.vaccine.2010.11.084

Systemic and mucosal humoral immune responses to gp140-adsorbed nanoparticles after intranasal immunization. (A) and (B) Kinetics of serum and vaginal IgG and IgA levels. Groups of 5 animals were immunized i.n. with 20 μg of gp140 either free or adsorbed to YC-NaMA NP. Serum and vaginal lavages were obtained before each immunization and 21 days after the last immunization, and tested for specific-gp140 IgG and IgA end-point titers by ELISA. Means were compared by two-way ANOVA. *** P
Figure Legend Snippet: Systemic and mucosal humoral immune responses to gp140-adsorbed nanoparticles after intranasal immunization. (A) and (B) Kinetics of serum and vaginal IgG and IgA levels. Groups of 5 animals were immunized i.n. with 20 μg of gp140 either free or adsorbed to YC-NaMA NP. Serum and vaginal lavages were obtained before each immunization and 21 days after the last immunization, and tested for specific-gp140 IgG and IgA end-point titers by ELISA. Means were compared by two-way ANOVA. *** P

Techniques Used: Enzyme-linked Immunosorbent Assay

In vivo humoral immune responses to antigen-adsorbed nanoparticles. Kinetics of specific serum IgG after i.d. immunization with (A) TT and (B) gp140. Groups of 3 (TT) and 8 (gp140) mice were immunized three times with (A) 12.5 μg TT and (B) 20 μg gp140 either free or adsorbed to NP. Ag adsorbed to Alum was used as a positive control of immunization. Serum samples were obtained before each immunization and 30 days after the last immunization, and tested for IgG end-point titers by ELISA. Results shown are the mean ± SEM of one experiment (TT) and two aggregated experiments (gp140). Mean differences were analyzed by two-way ANOVA with Bonferroni posttest to compare means by pairs. * P
Figure Legend Snippet: In vivo humoral immune responses to antigen-adsorbed nanoparticles. Kinetics of specific serum IgG after i.d. immunization with (A) TT and (B) gp140. Groups of 3 (TT) and 8 (gp140) mice were immunized three times with (A) 12.5 μg TT and (B) 20 μg gp140 either free or adsorbed to NP. Ag adsorbed to Alum was used as a positive control of immunization. Serum samples were obtained before each immunization and 30 days after the last immunization, and tested for IgG end-point titers by ELISA. Results shown are the mean ± SEM of one experiment (TT) and two aggregated experiments (gp140). Mean differences were analyzed by two-way ANOVA with Bonferroni posttest to compare means by pairs. * P

Techniques Used: In Vivo, Mouse Assay, Positive Control, Enzyme-linked Immunosorbent Assay

33) Product Images from "The volatile anesthetic isoflurane induces ecto-5?-nucleotidase (CD73) to protect against renal ischemia and reperfusion injury"

Article Title: The volatile anesthetic isoflurane induces ecto-5?-nucleotidase (CD73) to protect against renal ischemia and reperfusion injury

Journal: Kidney international

doi: 10.1038/ki.2013.43

TGF-β1 is responsible for isoflurane-mediated induction of CD73 and adenosine generation A and B. CD73 mRNA (RT-PCR, A) and protein (immunoblotting, B) expression in HK-2 cells treated with 2.5% isoflurane for 6 or 16 hr (N=4–6). Representative images (top) and band intensity quantifications (bottom) expressed as fold increases in CD73 expression over carrier gas and IgG isotype antibody treated controls. C. Adenosine levels in cell culture media (top) and CD73 activity (bottom) in HK-2 cells treated with 2.5% isoflurane for 6 hr (N=6). D and E. CD73 mRNA (RT-PCR, A) and protein (immunoblotting, B) expression in primary culture of mouse proximal tubule cells treated with 2.5% isoflurane for 6 or 16 hr (N=3). Representative images (top) and band intensity quantifications (bottom) expressed as fold increases in CD73 expression over carrier gas and IgG isotype antibody treated controls. GAPDH mRNA and b-actin protein expression were also quantified to normalize lane loading. *P
Figure Legend Snippet: TGF-β1 is responsible for isoflurane-mediated induction of CD73 and adenosine generation A and B. CD73 mRNA (RT-PCR, A) and protein (immunoblotting, B) expression in HK-2 cells treated with 2.5% isoflurane for 6 or 16 hr (N=4–6). Representative images (top) and band intensity quantifications (bottom) expressed as fold increases in CD73 expression over carrier gas and IgG isotype antibody treated controls. C. Adenosine levels in cell culture media (top) and CD73 activity (bottom) in HK-2 cells treated with 2.5% isoflurane for 6 hr (N=6). D and E. CD73 mRNA (RT-PCR, A) and protein (immunoblotting, B) expression in primary culture of mouse proximal tubule cells treated with 2.5% isoflurane for 6 or 16 hr (N=3). Representative images (top) and band intensity quantifications (bottom) expressed as fold increases in CD73 expression over carrier gas and IgG isotype antibody treated controls. GAPDH mRNA and b-actin protein expression were also quantified to normalize lane loading. *P

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture, Activity Assay

34) Product Images from "Experimental Validation of Multi-Epitope Peptides Including Promising MHC Class I- and II-Restricted Epitopes of Four Known Leishmania infantum Proteins"

Article Title: Experimental Validation of Multi-Epitope Peptides Including Promising MHC Class I- and II-Restricted Epitopes of Four Known Leishmania infantum Proteins

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2014.00268

Multi-epitope peptide-specific antibody production . BALB/c mice ( n = 9/group) immunized either with individual peptide emulsified in CFA/IFA or PBS alone, were bled 15 days post third immunization and sera were separated. (A,B) total IgG Abs, and (C) IgG1 and IgG2a Abs against each peptide were assessed by ELISA. The results are expressed as OD 450 ± SD. Significant differences between groups of mice immunized with each synthetic peptide emulsified in CFA/IFA and the group of mice immunized with CFA/IFA alone are indicated by * ( P
Figure Legend Snippet: Multi-epitope peptide-specific antibody production . BALB/c mice ( n = 9/group) immunized either with individual peptide emulsified in CFA/IFA or PBS alone, were bled 15 days post third immunization and sera were separated. (A,B) total IgG Abs, and (C) IgG1 and IgG2a Abs against each peptide were assessed by ELISA. The results are expressed as OD 450 ± SD. Significant differences between groups of mice immunized with each synthetic peptide emulsified in CFA/IFA and the group of mice immunized with CFA/IFA alone are indicated by * ( P

Techniques Used: Mouse Assay, Immunofluorescence, Enzyme-linked Immunosorbent Assay

35) Product Images from "Oral vaccination with a recombinant Salmonella vaccine vector provokes systemic HIV-1 subtype C Gag-specific CD4+ Th1 and Th2 cell immune responses in mice"

Article Title: Oral vaccination with a recombinant Salmonella vaccine vector provokes systemic HIV-1 subtype C Gag-specific CD4+ Th1 and Th2 cell immune responses in mice

Journal: Virology Journal

doi: 10.1186/1743-422X-6-87

HIV-1 subtype C Gag-specific serum IgG responses in mice vaccinated with recombinant Salmonella vaccine vector . Groups of mice (5 per group) were vaccinated with live recombinant Salmonella vaccine that expressed HIV-1 Subtype C Gag (aroC+Gag) or an antigen-negative Salmonella control vaccine (aroC+pGEM) as indicated in Figure 2. Serum (pooled from 5 mice per group) was isolated from blood taken before each vaccination on day 0, 28 and 56 and just before sacrifice on day 84. (A) The HIV-1 Gag-specific IgG for each group of mice with a 1/100 serum dilution. The data are the ratio of the OD 405 nm for vaccinated mice and the OD 405 nm for the day 0 serum (pre-bleed). (B) The HIV-1 Gag-specific IgG1 and IgG2a were measured in serum of each group of mice on Day 84 with a 1/100 serum dilution. Each bar represents the mean OD 405 nm value. Differences in antibody responses between vaccine groups at different time points were analyzed by a two-sample student's t-test and a p
Figure Legend Snippet: HIV-1 subtype C Gag-specific serum IgG responses in mice vaccinated with recombinant Salmonella vaccine vector . Groups of mice (5 per group) were vaccinated with live recombinant Salmonella vaccine that expressed HIV-1 Subtype C Gag (aroC+Gag) or an antigen-negative Salmonella control vaccine (aroC+pGEM) as indicated in Figure 2. Serum (pooled from 5 mice per group) was isolated from blood taken before each vaccination on day 0, 28 and 56 and just before sacrifice on day 84. (A) The HIV-1 Gag-specific IgG for each group of mice with a 1/100 serum dilution. The data are the ratio of the OD 405 nm for vaccinated mice and the OD 405 nm for the day 0 serum (pre-bleed). (B) The HIV-1 Gag-specific IgG1 and IgG2a were measured in serum of each group of mice on Day 84 with a 1/100 serum dilution. Each bar represents the mean OD 405 nm value. Differences in antibody responses between vaccine groups at different time points were analyzed by a two-sample student's t-test and a p

Techniques Used: Mouse Assay, Recombinant, Plasmid Preparation, Isolation

36) Product Images from "A 21.6 kDa tegumental protein of Clonorchis sinensis induces a Th1/Th2 mixed immune response in mice"

Article Title: A 21.6 kDa tegumental protein of Clonorchis sinensis induces a Th1/Th2 mixed immune response in mice

Journal: Immunity, Inflammation and Disease

doi: 10.1002/iid3.235

rCsTegu21.6 induced specific antibody production in C57BL/6 mice. rCsTegu21.6 (with or without alum adjuvant (Alum) or Freund's adjuvant (FA) was used to immunize C57BL/6 mice ( n = 5 per group) three times after two‐week intervals. Total IgG (A) and their isotypes (B) were detected in the immunized serum by ELISA. The results are shown as the mean ± standard error (SEM). * P
Figure Legend Snippet: rCsTegu21.6 induced specific antibody production in C57BL/6 mice. rCsTegu21.6 (with or without alum adjuvant (Alum) or Freund's adjuvant (FA) was used to immunize C57BL/6 mice ( n = 5 per group) three times after two‐week intervals. Total IgG (A) and their isotypes (B) were detected in the immunized serum by ELISA. The results are shown as the mean ± standard error (SEM). * P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

37) Product Images from "Immunization with recombinant enolase of Sporothrix spp. (rSsEno) confers effective protection against sporotrichosis in mice"

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

Journal: Scientific Reports

doi: 10.1038/s41598-019-53135-z

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

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

38) Product Images from "Gene Therapy Induces Antigen-Specific Tolerance in Experimental Collagen-Induced Arthritis"

Article Title: Gene Therapy Induces Antigen-Specific Tolerance in Experimental Collagen-Induced Arthritis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0154630

Development of arthritis and B cell responses in the novel model of tolerance to collagen-induced arthritis. Severity (A) as well as incidence of arthritis (B) were determined by macroscopical examination (LNT-Ctrl n = 25 (day 0–42), n = 17 (day 43–49) LNT-CII n = 24 (day 0–42), n = 11 (day 43–49)). The experiments have been repeated five times and the graph shows the pooled results from three experiments (C) Histopathological examination of synovitis and bone/cartilage erosivity at termination of two pooled experiments (days 42–49). (D) Histological photos from an LNT-Ctrl and an LNT-CII mouse at day 42 after CII immunization, B = bone, C = cartilage, S = synovium. Scale bar 100 μm. (E) Development of CII specific IgG antibodies measured by ELISA in serum at indicated time points during CIA (n = 3-6/group). (F) Serum levels of CII-specific IgG and subclasses IgG1, IgG2a and IgG2b at days 42–49. Experiments in Fig E and F have been performed at least twice. Statistical analysis was performed by linear regression in (A, D), logistic regression in (B) and Mann-Whitney U-test in (C and E), mean±SEM.
Figure Legend Snippet: Development of arthritis and B cell responses in the novel model of tolerance to collagen-induced arthritis. Severity (A) as well as incidence of arthritis (B) were determined by macroscopical examination (LNT-Ctrl n = 25 (day 0–42), n = 17 (day 43–49) LNT-CII n = 24 (day 0–42), n = 11 (day 43–49)). The experiments have been repeated five times and the graph shows the pooled results from three experiments (C) Histopathological examination of synovitis and bone/cartilage erosivity at termination of two pooled experiments (days 42–49). (D) Histological photos from an LNT-Ctrl and an LNT-CII mouse at day 42 after CII immunization, B = bone, C = cartilage, S = synovium. Scale bar 100 μm. (E) Development of CII specific IgG antibodies measured by ELISA in serum at indicated time points during CIA (n = 3-6/group). (F) Serum levels of CII-specific IgG and subclasses IgG1, IgG2a and IgG2b at days 42–49. Experiments in Fig E and F have been performed at least twice. Statistical analysis was performed by linear regression in (A, D), logistic regression in (B) and Mann-Whitney U-test in (C and E), mean±SEM.

Techniques Used: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

39) Product Images from "Role of T3SS-1 SipD Protein in Protecting Mice against Non-typhoidal Salmonella Typhimurium"

Article Title: Role of T3SS-1 SipD Protein in Protecting Mice against Non-typhoidal Salmonella Typhimurium

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0005207

IgG (2a +2b) / IgG 1 ratio after PrgI (left) and SipD (right) immunizations. Mice immunized with PrgI or SipD separately are represented on panel A and those receiving both PrgI and SipD on panel B. Data represent mean and the standard errors (SEM) from 14–16 mice per group. [°: indicates immunogen injected; *: indicates biotinylated recombinant protein].
Figure Legend Snippet: IgG (2a +2b) / IgG 1 ratio after PrgI (left) and SipD (right) immunizations. Mice immunized with PrgI or SipD separately are represented on panel A and those receiving both PrgI and SipD on panel B. Data represent mean and the standard errors (SEM) from 14–16 mice per group. [°: indicates immunogen injected; *: indicates biotinylated recombinant protein].

Techniques Used: Mouse Assay, Injection, Recombinant

40) Product Images from "CD4+ T Lymphocytes Are Critical Mediators of Tumor Immunity to Simian Virus 40 Large Tumor Antigen Induced by Vaccination with Plasmid DNA ▿"

Article Title: CD4+ T Lymphocytes Are Critical Mediators of Tumor Immunity to Simian Virus 40 Large Tumor Antigen Induced by Vaccination with Plasmid DNA ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00543-11

Immunization with pCVM-Tag induces a mixed IgG subtype distribution of antibodies to SV40 Tag.
Figure Legend Snippet: Immunization with pCVM-Tag induces a mixed IgG subtype distribution of antibodies to SV40 Tag.

Techniques Used:

Related Articles

Incubation:

Article Title: Collagen epitope expression on B cells is sufficient to confer tolerance to collagen-induced arthritis
Article Snippet: .. Serum samples were diluted (1/7500, 1/22,500, 1/67,500 and 1/202,500) and after incubation CII-specific IgG was detected by biotinylated rat anti-mouse IgG, IgG1, IgG2a or IgG2b at 0.5 μg/ml (Serotec, Oxford, UK) or biotinylated (Fab)2 goat anti-mouse IgM (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). ..

Article Title: Oral antigen exposure in newborn piglets circumvents induction of oral tolerance in response to intraperitoneal vaccination in later life
Article Snippet: .. Wells were washed again with distilled H2 O and mouse anti-porcine IgA (Ab Serotec, Raleigh, NC, #MCA 638, 1/300), mouse anti-porcine IgG1 (Ab Serotec, #MCA 635, 1/600), mouse anti-porcine IgG2 (Ab Serotec, #MCA 636, 1/300), or mouse anti-porcine IgM (Ab Serotec, #MCA 637, 1/100) were added to the wells in a 100 μL volume and incubated for one hour at RT. .. Wells were washed again with dH2 O and goat anti-mouse IgG (H + L) alkaline phosphatase conjugated (KPL, Gaithersburg, MD, USA, #075-1806, 1/5000) was added to each well at 100 μL/well followed by incubation for one hour at room temperature (RT).

Article Title: Differential Pro-Inflammatory Responses of Astrocytes and Microglia Involve STAT3 Activation in Response to 1800 MHz Radiofrequency Fields
Article Snippet: .. Then, the cells were incubated with rat anti-mouse monoclonal antibody CD11b (1∶100; AbD Serotec, Oxford, UK) or rat IgG2b isotype control (1∶100; AbD Serotec) for 30 min at 4°C. .. Following three washes with staining buffer, the cells were then incubated with fluorescently labeled rabbit anti-rat IgG (1∶100; Invitrogen, Carlsbad, CA, USA) for 30 min at 4°C in the dark.

Article Title: A miR-335/COX-2/PTEN axis regulates the secretory phenotype of senescent cancer-associated fibroblasts
Article Snippet: .. For blockade of secreted MCP-1, CM was incubated with either anti-MCP-1 antibody (20 μγ/ml, subtype IgG, R & D: MAB679) or isotype IgG1 and IgG2 controls (Serotec: MCA1209, Invitrogen: MG2b00) 1 h preceding chemotaxis assay. ..

other:

Article Title: Adjuvanted poly(lactic-co-glycolic) acid nanoparticle-entrapped inactivated porcine reproductive and respiratory syndrome virus vaccine elicits cross-protective immune response in pigs
Article Snippet: Briefly killed semipurified PRRSV (MN184) Ags (5 μg/mL)-coated plates were blocked and plated with a serial tenfold dilution of plasma samples, and the plate-bound virus-specific IgG1 and IgG2 were detected using mouse antipig IgG1 and IgG2 (AbD Serotec; Bio-Rad Laboratories, Hercules, CA, USA) (1:250 dilution), respectively.

Chemotaxis Assay:

Article Title: A miR-335/COX-2/PTEN axis regulates the secretory phenotype of senescent cancer-associated fibroblasts
Article Snippet: .. For blockade of secreted MCP-1, CM was incubated with either anti-MCP-1 antibody (20 μγ/ml, subtype IgG, R & D: MAB679) or isotype IgG1 and IgG2 controls (Serotec: MCA1209, Invitrogen: MG2b00) 1 h preceding chemotaxis assay. ..

Marker:

Article Title: Preconditioned or IL4-Secreting Mesenchymal Stem Cells Enhanced Osteogenesis at Different Stages
Article Snippet: .. Immunofluorescent staining Macrophages marker F4/80 (1 μg/mL, Alexa Fluor 647-conjugated, monoclonal rat anti-mouse IgG2b; Bio-Rad) and their polarization markers, including CD206 (1 μg/mL, allophycocyanin-conjugated, monoclonal rat anti-mouse IgG2a κ; BioLegend, San Diego, CA), Arginase 1 (5 μg/mL, unconjugated, polyclonal rabbit anti-mouse IgG; Abcam, Cambridge, United Kingdom) were stained as previously described. .. The images were captured using a fluorescence microscope (Axio Observer 3.1; Zeiss, Oberkochen, Germany) in three randomly selected fields of view, and the signals of each marker were quantified by using NIH ImageJ.

Staining:

Article Title: Preconditioned or IL4-Secreting Mesenchymal Stem Cells Enhanced Osteogenesis at Different Stages
Article Snippet: .. Immunofluorescent staining Macrophages marker F4/80 (1 μg/mL, Alexa Fluor 647-conjugated, monoclonal rat anti-mouse IgG2b; Bio-Rad) and their polarization markers, including CD206 (1 μg/mL, allophycocyanin-conjugated, monoclonal rat anti-mouse IgG2a κ; BioLegend, San Diego, CA), Arginase 1 (5 μg/mL, unconjugated, polyclonal rabbit anti-mouse IgG; Abcam, Cambridge, United Kingdom) were stained as previously described. .. The images were captured using a fluorescence microscope (Axio Observer 3.1; Zeiss, Oberkochen, Germany) in three randomly selected fields of view, and the signals of each marker were quantified by using NIH ImageJ.

Binding Assay:

Article Title: Trypanosoma cruzi infection induces a massive extrafollicular and follicular splenic B-cell response which is a high source of non-parasite-specific antibodies
Article Snippet: .. The following reagents were used: rat anti-mouse CD3 (KT3) and rat anti-mouse IgM (LO MM 9; Serotec, Oxford, UK); rat anti-mouse syndecan-1 (CD138, 09341D; BD PharMingen, Oxford, UK); rat anti-mouse IgG1, IgG2a, IgG2b and IgG3 (Serotec, UK); biotinylated peanut agglutinin (PNA; Vector Laboratories, Burlingame, CA); rabbit anti-mouse Ki67 (a gift from Johannes Gerdes, Borstel, Germany), swine anti-rabbit immunoglobulin, rabbit anti-horseradish peroxidase (HRP) complexes, biotinylated rabbit anti rat immunoglobulin and alkaline phosphatase-labelled StreptABC complex (Dako, Cambridgeshire, UK), sheep anti-mouse IgD, HRP-labelled donkey anti-sheep/goat immunoglobulin (The Binding Site, Birmingham, UK). ..

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    Bio-Rad secondary antibodies against bovine igg1
    Titrated IgM, <t>IgG1,</t> and IgG2 antibody response in animals vaccinated with salivary gland extract (SGE), midgut extract (ME) or a combination of both SGE and ME (SGE+ME). Plates were coated with 0.5 μg/mL SGE (A,B) or ME (C,D) , respectively.
    Secondary Antibodies Against Bovine Igg1, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad human anti cenp a igg cenp a derived peptide 1 17
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    Human Anti Cenp A Igg Cenp A Derived Peptide 1 17, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human anti cenp a igg cenp a derived peptide 1 17/product/Bio-Rad
    Average 85 stars, based on 1 article reviews
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    Bio-Rad novalisa sars cov 2 covid 19 igg
    Evolution of the clinical sensitivity over 8 weeks according to the manufacturer's cut‐off and the adapted cut‐off for the <t>NovaLisa</t> SARS‐CoV‐2 (COVID‐19) IgG (A), IgA (B), and IgM (C) tests and (D) for the Platelia SARS‐CoV‐2 Total Ab method (Bio‐Rad). IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2
    Novalisa Sars Cov 2 Covid 19 Igg, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/novalisa sars cov 2 covid 19 igg/product/Bio-Rad
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    novalisa sars cov 2 covid 19 igg - by Bioz Stars, 2020-09
    84/100 stars
      Buy from Supplier

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    Titrated IgM, IgG1, and IgG2 antibody response in animals vaccinated with salivary gland extract (SGE), midgut extract (ME) or a combination of both SGE and ME (SGE+ME). Plates were coated with 0.5 μg/mL SGE (A,B) or ME (C,D) , respectively.

    Journal: Frontiers in Physiology

    Article Title: Preliminary Evaluation of Tick Protein Extracts and Recombinant Ferritin 2 as Anti-tick Vaccines Targeting Ixodes ricinus in Cattle

    doi: 10.3389/fphys.2018.01696

    Figure Lengend Snippet: Titrated IgM, IgG1, and IgG2 antibody response in animals vaccinated with salivary gland extract (SGE), midgut extract (ME) or a combination of both SGE and ME (SGE+ME). Plates were coated with 0.5 μg/mL SGE (A,B) or ME (C,D) , respectively.

    Article Snippet: The plates were then washed, blocked with 1% fetal bovine serum in PBS, incubated with the sera dilutions followed by specific secondary antibodies against bovine IgG1, IgG2, and IgM conjugated with horseradish peroxidase (Bio-Rad).

    Techniques:

    Affinity chromatography of non-modified and FITC-modified pIgG mix on DNA-cellulose. (–) and (–), absorbance of IgGs at 280 nm before and after modification of IgGs with FITC, respectively; the bars correspond to the relative fluorescence of FITC-IgG mix fractions (A). Analysis of a relative efficiency of specific-molecule exchange under different conditions between non-modified IgG mix and FITC-IgG mix having different affinity for DNA-cellulose (B–D). Before chromatography, the IgG mix eluted from DNA-cellulose by 0.15 M NaCl (0.15 M-IgG mix ) were incubated with FITC-IgG mix eluted by 1.5 M NaCl (1.5 M-FITC-IgG mix ) in the presence of TBS and GSH (B); 0.5 M-IgG mix and 1.5 M-FITC-IgG mix (C) or 0.5 M-FITC-IgG mix +1.5 M-IgG mix (D) were incubated in the presence of TBS containing GSH and human milk plasma.

    Journal: PLoS ONE

    Article Title: Human Milk IgGs Contain Various Combinations of Different Antigen-Binding Sites Resulting in Multiple Variants of their Bispecificity

    doi: 10.1371/journal.pone.0042942

    Figure Lengend Snippet: Affinity chromatography of non-modified and FITC-modified pIgG mix on DNA-cellulose. (–) and (–), absorbance of IgGs at 280 nm before and after modification of IgGs with FITC, respectively; the bars correspond to the relative fluorescence of FITC-IgG mix fractions (A). Analysis of a relative efficiency of specific-molecule exchange under different conditions between non-modified IgG mix and FITC-IgG mix having different affinity for DNA-cellulose (B–D). Before chromatography, the IgG mix eluted from DNA-cellulose by 0.15 M NaCl (0.15 M-IgG mix ) were incubated with FITC-IgG mix eluted by 1.5 M NaCl (1.5 M-FITC-IgG mix ) in the presence of TBS and GSH (B); 0.5 M-IgG mix and 1.5 M-FITC-IgG mix (C) or 0.5 M-FITC-IgG mix +1.5 M-IgG mix (D) were incubated in the presence of TBS containing GSH and human milk plasma.

    Article Snippet: The fluorescence of FITC-IgGs was analyzed using a PharosFX imaging system (Bio-Rad; fluorophores mode, FITC, high sample intensity).

    Techniques: Affinity Chromatography, Modification, Fluorescence, Chromatography, Incubation

    Reactivity of affinity-purified anti-Ap 1-17 Abs from 3 SSc patients. ( A. ) Recombinant human CENP-B and CENP-A proteins were loaded on alternative lanes (200 ng/lane) of a 12.5% SDS mini-gel under non-reducing conditions, transferred to a PVDF filter, and incubated for 3 h with affinity-purified anti-Ap 1-17 Abs from pt1, pt4 and pt14. Serum from pt1 and IVIG were used as controls. Bound Abs were detected using HRP-conjugated goat anti-human IgG and diaminobenzidine substrate solution. ( B. ) Centromere staining of pt1, pt4, pt8 and pt14 anti-Ap 1-17 IgG and of their corresponding serum (1∶100 dilution) to fixed permeabilized HeLa cells. Bound IgG was revealed by fluorescence staining with FITC-conjugated anti-human IgG (Fc portion). Cells were examined with a Nikon confocal microscope and a CCD camera (Nikon digital sight DS-U1), using a 60× Plan Apo VC objective.

    Journal: PLoS ONE

    Article Title: Autoantibodies Recognizing the Amino Terminal 1-17 Segment of CENP-A Display Unique Specificities in Systemic Sclerosis

    doi: 10.1371/journal.pone.0061453

    Figure Lengend Snippet: Reactivity of affinity-purified anti-Ap 1-17 Abs from 3 SSc patients. ( A. ) Recombinant human CENP-B and CENP-A proteins were loaded on alternative lanes (200 ng/lane) of a 12.5% SDS mini-gel under non-reducing conditions, transferred to a PVDF filter, and incubated for 3 h with affinity-purified anti-Ap 1-17 Abs from pt1, pt4 and pt14. Serum from pt1 and IVIG were used as controls. Bound Abs were detected using HRP-conjugated goat anti-human IgG and diaminobenzidine substrate solution. ( B. ) Centromere staining of pt1, pt4, pt8 and pt14 anti-Ap 1-17 IgG and of their corresponding serum (1∶100 dilution) to fixed permeabilized HeLa cells. Bound IgG was revealed by fluorescence staining with FITC-conjugated anti-human IgG (Fc portion). Cells were examined with a Nikon confocal microscope and a CCD camera (Nikon digital sight DS-U1), using a 60× Plan Apo VC objective.

    Article Snippet: Affinity purification of human anti-CENP-A IgG CENP-A-derived peptide 1-17 (Ap1-17 ; 1 MGPRRRSRKPEAPRRRS17 ) was conjugated to AffiGel 15 (Bio-Rad Laboratories, Hercules, CA, USA) at a concentration of 2 mg/ml resin following the manufacturer's instructions and used to purify human anti-CENP-A IgG as previously described .

    Techniques: Affinity Purification, Recombinant, Incubation, Staining, Fluorescence, Microscopy

    Specificity of anti-Ap 1-17 IgG for CENP-A documented by human recombinant CENP-A inhibition of anti-Ap 1-17 IgG binding to KLH-Ap 1-17 . Anti-Ap 1-17 Abs from pt4 (A) and pt14 (B) were diluted in PBS-T20 at the lowest concentration giving 80%–100% of maximal A 490 in binding assay, and pre-incubated with an equal volume of PBS containing 2-fold serial dilutions of CENP-A (○), CENP-B (•), and CENP-B-derived peptide CBp 1-13 (▪). Following a 2-h incubation, the mixture was added to microtiter plate wells coated with KLH-Ap 1-17 . After a 4-h incubation and three washes, bound IgG was detected with HRP-conjugated anti-human IgG (Fc portion) and o -phenylenediamine. Inhibition by Ap 1-17 peptide (□) and by Qp-1a (X) were included as positive and negative controls, respectively. Results are expressed as percentage of binding inhibition. The data are representative of 2 experiments.

    Journal: PLoS ONE

    Article Title: Autoantibodies Recognizing the Amino Terminal 1-17 Segment of CENP-A Display Unique Specificities in Systemic Sclerosis

    doi: 10.1371/journal.pone.0061453

    Figure Lengend Snippet: Specificity of anti-Ap 1-17 IgG for CENP-A documented by human recombinant CENP-A inhibition of anti-Ap 1-17 IgG binding to KLH-Ap 1-17 . Anti-Ap 1-17 Abs from pt4 (A) and pt14 (B) were diluted in PBS-T20 at the lowest concentration giving 80%–100% of maximal A 490 in binding assay, and pre-incubated with an equal volume of PBS containing 2-fold serial dilutions of CENP-A (○), CENP-B (•), and CENP-B-derived peptide CBp 1-13 (▪). Following a 2-h incubation, the mixture was added to microtiter plate wells coated with KLH-Ap 1-17 . After a 4-h incubation and three washes, bound IgG was detected with HRP-conjugated anti-human IgG (Fc portion) and o -phenylenediamine. Inhibition by Ap 1-17 peptide (□) and by Qp-1a (X) were included as positive and negative controls, respectively. Results are expressed as percentage of binding inhibition. The data are representative of 2 experiments.

    Article Snippet: Affinity purification of human anti-CENP-A IgG CENP-A-derived peptide 1-17 (Ap1-17 ; 1 MGPRRRSRKPEAPRRRS17 ) was conjugated to AffiGel 15 (Bio-Rad Laboratories, Hercules, CA, USA) at a concentration of 2 mg/ml resin following the manufacturer's instructions and used to purify human anti-CENP-A IgG as previously described .

    Techniques: Recombinant, Inhibition, Binding Assay, Concentration Assay, Incubation, Derivative Assay

    Evolution of the clinical sensitivity over 8 weeks according to the manufacturer's cut‐off and the adapted cut‐off for the NovaLisa SARS‐CoV‐2 (COVID‐19) IgG (A), IgA (B), and IgM (C) tests and (D) for the Platelia SARS‐CoV‐2 Total Ab method (Bio‐Rad). IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2

    Journal: Journal of Medical Virology

    Article Title: Analytical and clinical validation of an ELISA for specific SARS‐CoV‐2 IgG, IgA, and IgM antibodies, et al. Analytical and clinical validation of an ELISA for specific SARS‐CoV‐2 IgG, IgA and IgM antibodies

    doi: 10.1002/jmv.26303

    Figure Lengend Snippet: Evolution of the clinical sensitivity over 8 weeks according to the manufacturer's cut‐off and the adapted cut‐off for the NovaLisa SARS‐CoV‐2 (COVID‐19) IgG (A), IgA (B), and IgM (C) tests and (D) for the Platelia SARS‐CoV‐2 Total Ab method (Bio‐Rad). IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2

    Article Snippet: The adapted cut‐off of the NovaLisa SARS‐CoV‐2 (COVID‐19) IgG, IgA, and IgM test (NovaTec) and the Platelia SARS‐CoV‐2 Total Ab method (Bio‐Rad) was determined using receiver operator characteristic (ROC) curves at 14 ± 2 days post RT‐qPCR.

    Techniques: