rat anti mouse igg  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99

    Structured Review

    Thermo Fisher rat anti mouse igg
    Influenza virus–specific antibody response in AID −/− mice. Mice of each genotype, AID −/− or AID +/− , were intranasally inoculated with 1 PFU mPR8 and 42 d later they were challenged intranasally with a lethal dose (1,000 PFU) of mPR8 virus (arrows). Blood was taken at the indicated time points and virus-specific titers of total antibodies, <t>IgM</t> and <t>IgG,</t> and virus-neutralizing antibody titers in the sera were determined as described in Materials and Methods. Symbols and error bars represent means and standard deviations for three to five mice per group. The limit of detection in this assay was 4 2.6 . Titers
    Rat Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5660 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rat anti mouse igg/product/Thermo Fisher
    Average 99 stars, based on 5660 article reviews
    Price from $9.99 to $1999.99
    rat anti mouse igg - by Bioz Stars, 2021-01
    99/100 stars

    Images

    1) Product Images from "Unmutated Immunoglobulin M Can Protect Mice from Death by Influenza Virus Infection"

    Article Title: Unmutated Immunoglobulin M Can Protect Mice from Death by Influenza Virus Infection

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20021457

    Influenza virus–specific antibody response in AID −/− mice. Mice of each genotype, AID −/− or AID +/− , were intranasally inoculated with 1 PFU mPR8 and 42 d later they were challenged intranasally with a lethal dose (1,000 PFU) of mPR8 virus (arrows). Blood was taken at the indicated time points and virus-specific titers of total antibodies, IgM and IgG, and virus-neutralizing antibody titers in the sera were determined as described in Materials and Methods. Symbols and error bars represent means and standard deviations for three to five mice per group. The limit of detection in this assay was 4 2.6 . Titers
    Figure Legend Snippet: Influenza virus–specific antibody response in AID −/− mice. Mice of each genotype, AID −/− or AID +/− , were intranasally inoculated with 1 PFU mPR8 and 42 d later they were challenged intranasally with a lethal dose (1,000 PFU) of mPR8 virus (arrows). Blood was taken at the indicated time points and virus-specific titers of total antibodies, IgM and IgG, and virus-neutralizing antibody titers in the sera were determined as described in Materials and Methods. Symbols and error bars represent means and standard deviations for three to five mice per group. The limit of detection in this assay was 4 2.6 . Titers

    Techniques Used: Mouse Assay

    2) Product Images from "Independent modes of disease repair by AIM protein distinguished in AIM-felinized mice"

    Article Title: Independent modes of disease repair by AIM protein distinguished in AIM-felinized mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31580-6

    States of inflammation and fibrosis in the liver in the absence of serum IgM-free AIM. ( a ) The mRNA levels of various genes responsive to different types of stresses (i.e., endoplasmic reticulum, mitochondrial, or oxidative stress) addressed by qPCR using RNA from the whole liver of AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 5–6 for 12-week HFD per group). Error bar indicates the SEM. (b,c) The mRNA levels of inflammatory cytokine genes in the liver, as assessed by qPCR using the RNA as in ( a ). (d ) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsμ mice fed an HFD for 12 weeks stained with Sirius red Stain. Scale bars, 100 μm. (e) The mRNA levels of fibrogenetic genes in the liver, as assessed by qPCR using the same RNA as in ( a ). In (a – c) and (e) , *is used to represent the statistical significance between the values of each mouse strain group within the same period, whereas # is attached to the bar of 12 w when the value of 12 w was significantly changed compared with that of 0 w in the same mouse strain group.
    Figure Legend Snippet: States of inflammation and fibrosis in the liver in the absence of serum IgM-free AIM. ( a ) The mRNA levels of various genes responsive to different types of stresses (i.e., endoplasmic reticulum, mitochondrial, or oxidative stress) addressed by qPCR using RNA from the whole liver of AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 5–6 for 12-week HFD per group). Error bar indicates the SEM. (b,c) The mRNA levels of inflammatory cytokine genes in the liver, as assessed by qPCR using the RNA as in ( a ). (d ) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsμ mice fed an HFD for 12 weeks stained with Sirius red Stain. Scale bars, 100 μm. (e) The mRNA levels of fibrogenetic genes in the liver, as assessed by qPCR using the same RNA as in ( a ). In (a – c) and (e) , *is used to represent the statistical significance between the values of each mouse strain group within the same period, whereas # is attached to the bar of 12 w when the value of 12 w was significantly changed compared with that of 0 w in the same mouse strain group.

    Techniques Used: Real-time Polymerase Chain Reaction, Mouse Assay, Staining

    Effect of serum IgM-free AIM on obesity and liver steatosis. ( a,b) Weights from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM. (a) Body weights. (b ) Weights of epidydimal adipose tissues. (c) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Adipocyte sizes of 50 independent adipocytes in different areas were evaluated. Results are presented as average ± SEM (in μm 2 ). Scale bars, 100 μm. ( d ) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, and AIM −/− mice (fed an HFD for 12 weeks) stained for AIM (blue), F4/80 (macrophage marker; green), and IgM (red, WT and AIM −/− mice. See Supplementary Fig. 1 for AIM-felinized mice). Yellow arrows represent where IgM-free AIM signals exist, while red arrows indicate where AIM is co-stained with IgM. Scale bars, 100 μm. (e) The mRNA levels of AIM and F4/80 were assessed by qPCR using RNA isolated from epididymal fat in WT mice before or after being fed an HFD for 12 weeks. Values were normalized to those of GAPDH and presented as the expression relative to that of AIM from lean WT mice liver and of F4/80 from fat tissues before being fed an HFD (n = 4 per group). Error bar indicates the SEM. (f) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Scale bars, 100 µm. (g) Liver weights and TG contents from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM.
    Figure Legend Snippet: Effect of serum IgM-free AIM on obesity and liver steatosis. ( a,b) Weights from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM. (a) Body weights. (b ) Weights of epidydimal adipose tissues. (c) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Adipocyte sizes of 50 independent adipocytes in different areas were evaluated. Results are presented as average ± SEM (in μm 2 ). Scale bars, 100 μm. ( d ) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, and AIM −/− mice (fed an HFD for 12 weeks) stained for AIM (blue), F4/80 (macrophage marker; green), and IgM (red, WT and AIM −/− mice. See Supplementary Fig. 1 for AIM-felinized mice). Yellow arrows represent where IgM-free AIM signals exist, while red arrows indicate where AIM is co-stained with IgM. Scale bars, 100 μm. (e) The mRNA levels of AIM and F4/80 were assessed by qPCR using RNA isolated from epididymal fat in WT mice before or after being fed an HFD for 12 weeks. Values were normalized to those of GAPDH and presented as the expression relative to that of AIM from lean WT mice liver and of F4/80 from fat tissues before being fed an HFD (n = 4 per group). Error bar indicates the SEM. (f) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Scale bars, 100 µm. (g) Liver weights and TG contents from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM.

    Techniques Used: Mouse Assay, Staining, Marker, Real-time Polymerase Chain Reaction, Isolation, Expressing

    3) Product Images from "Suppression of Glomerulonephritis in NZB/NZW Lupus Prone Mice by Adoptive Transfer of Ex Vivo Expanded Regulatory T Cells"

    Article Title: Suppression of Glomerulonephritis in NZB/NZW Lupus Prone Mice by Adoptive Transfer of Ex Vivo Expanded Regulatory T Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006031

    Inhibition of immune complex renal damage in T reg treated mice. Typical kidney sections demonstrating less severe IgG and IgM deposition in mice receiving adoptive T reg transfer ( Figure 6A–B respectively) as compared to mice receiving either control therapy (T eff control mouse, Figure 6C–D ).
    Figure Legend Snippet: Inhibition of immune complex renal damage in T reg treated mice. Typical kidney sections demonstrating less severe IgG and IgM deposition in mice receiving adoptive T reg transfer ( Figure 6A–B respectively) as compared to mice receiving either control therapy (T eff control mouse, Figure 6C–D ).

    Techniques Used: Inhibition, Mouse Assay

    4) Product Images from "Direct effects of HP Acthar Gel® on human B lymphocyte activation in vitro"

    Article Title: Direct effects of HP Acthar Gel® on human B lymphocyte activation in vitro

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-015-0823-y

    Immunoglobulin production by activated human B lymphocytes in vitro is suppressed by Acthar. IgG ( top panel ) and IgM ( bottom panel ) were measured in supernatants from human peripheral B cells cultured for 6 days under basal conditions, with added IL-4/CD40L alone, or with IL-4/CD40L plus added Acthar at ACTH analog concentrations of approximately 0.124, 1.24, or 2.49 μM ( stepped bars ). Parallel cultures contained IL-4/CD40L plus matched volumes of placebo. Supernatants were harvested and assayed for IgG or IgM concentration and results analyzed by ANOVA (Kruskal–Wallis test, p
    Figure Legend Snippet: Immunoglobulin production by activated human B lymphocytes in vitro is suppressed by Acthar. IgG ( top panel ) and IgM ( bottom panel ) were measured in supernatants from human peripheral B cells cultured for 6 days under basal conditions, with added IL-4/CD40L alone, or with IL-4/CD40L plus added Acthar at ACTH analog concentrations of approximately 0.124, 1.24, or 2.49 μM ( stepped bars ). Parallel cultures contained IL-4/CD40L plus matched volumes of placebo. Supernatants were harvested and assayed for IgG or IgM concentration and results analyzed by ANOVA (Kruskal–Wallis test, p

    Techniques Used: In Vitro, Cell Culture, Concentration Assay

    5) Product Images from "A public antibody lineage that potently inhibits malaria infection by dual binding to the circumsporozoite protein"

    Article Title: A public antibody lineage that potently inhibits malaria infection by dual binding to the circumsporozoite protein

    Journal: Nature medicine

    doi: 10.1038/nm.4513

    Immunization with PfSPZ Vaccine induces robust antibody responses in malaria-exposed individuals a, Protocol of PfSPZ immunization of Tanzanian adults. b, Binding of serum IgM and IgG antibodies to PfSPZ. Median fluorescence intensity (MFI) values are for binding at a 1/1000 serum dilution (representative of n=2 independent experiments). Samples in red, black and blue are from protected (U, G, V, H), non-protected (NP) and placebo (C) volunteers, respectively. Results for donor W are not shown as this donor was immunized with a lower dose of PfSPZ. c, Staining of PfSPZ by serum from a European blood donor, serum from a protected individual (donor H) and a monoclonal antibody (MGU8) (representative of n=3 independent experiments). d, Dose-dependent binding of three representative antibodies to PfSPZ measured by flow cytometry (representative of n=2 independent experiments). e, Binding values of the panel of IgG monoclonal antibodies to PfSPZ (representative of n=2 independent experiments). The values indicate the concentration of antibody required to reach a 10,000 MFI. f, Number of PfSPZ-binding IgG and IgM monoclonal antibodies isolated from protected donors G and U. g, Number of mutations in the heavy chains of IgG (n=19 antibodies) and IgM (n=65 antibodies) isolated from the Tanzanian volunteers. These values were calculated by adding the number of VH and JH mutations. Results are shown as mean ± s.d.. A two-sided t-test was used to compare the number of mutations.
    Figure Legend Snippet: Immunization with PfSPZ Vaccine induces robust antibody responses in malaria-exposed individuals a, Protocol of PfSPZ immunization of Tanzanian adults. b, Binding of serum IgM and IgG antibodies to PfSPZ. Median fluorescence intensity (MFI) values are for binding at a 1/1000 serum dilution (representative of n=2 independent experiments). Samples in red, black and blue are from protected (U, G, V, H), non-protected (NP) and placebo (C) volunteers, respectively. Results for donor W are not shown as this donor was immunized with a lower dose of PfSPZ. c, Staining of PfSPZ by serum from a European blood donor, serum from a protected individual (donor H) and a monoclonal antibody (MGU8) (representative of n=3 independent experiments). d, Dose-dependent binding of three representative antibodies to PfSPZ measured by flow cytometry (representative of n=2 independent experiments). e, Binding values of the panel of IgG monoclonal antibodies to PfSPZ (representative of n=2 independent experiments). The values indicate the concentration of antibody required to reach a 10,000 MFI. f, Number of PfSPZ-binding IgG and IgM monoclonal antibodies isolated from protected donors G and U. g, Number of mutations in the heavy chains of IgG (n=19 antibodies) and IgM (n=65 antibodies) isolated from the Tanzanian volunteers. These values were calculated by adding the number of VH and JH mutations. Results are shown as mean ± s.d.. A two-sided t-test was used to compare the number of mutations.

    Techniques Used: Binding Assay, Fluorescence, Staining, Flow Cytometry, Cytometry, Concentration Assay, Isolation

    6) Product Images from "Biomimetic soft fibrous hydrogels for contractile and pharmacologically responsive smooth muscle"

    Article Title: Biomimetic soft fibrous hydrogels for contractile and pharmacologically responsive smooth muscle

    Journal: Acta biomaterialia

    doi: 10.1016/j.actbio.2018.05.015

    Soft fibre networks lead to lower cell proliferation. (A) Quantification of the percentage of proliferative cells, Ki-67-positive cells on three representative protein combinations after 1 day post-seeding. * stiff versus soft for p
    Figure Legend Snippet: Soft fibre networks lead to lower cell proliferation. (A) Quantification of the percentage of proliferative cells, Ki-67-positive cells on three representative protein combinations after 1 day post-seeding. * stiff versus soft for p

    Techniques Used:

    7) Product Images from "CXCL12 induces migration of oligodendrocyte precursor cells through the CXCR4-activated MEK/ERK and PI3K/AKT pathways"

    Article Title: CXCL12 induces migration of oligodendrocyte precursor cells through the CXCR4-activated MEK/ERK and PI3K/AKT pathways

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2018.9444

    Isolation and identification of OPCs. (A) Immunostaining demonstrated that OPCs were positive for NG2. (B) Immunostaining demonstrated that OPCs were positive for PDGFR-α. (C) Immunostaining showed that the differentiated OPCs were positive for O4. (D) Immunostaining showed that the differentiated OPCs were positive for MBP. Nuclei were stained with DAPI (blue). NG2, neural/glial antigen 2; PDGFR-α, platelet-derived growth factor receptor-α; OPCs, oligodendrocyte precursor cells; O4, oligodendrocyte marker O4; MBP, myelin basic protein.
    Figure Legend Snippet: Isolation and identification of OPCs. (A) Immunostaining demonstrated that OPCs were positive for NG2. (B) Immunostaining demonstrated that OPCs were positive for PDGFR-α. (C) Immunostaining showed that the differentiated OPCs were positive for O4. (D) Immunostaining showed that the differentiated OPCs were positive for MBP. Nuclei were stained with DAPI (blue). NG2, neural/glial antigen 2; PDGFR-α, platelet-derived growth factor receptor-α; OPCs, oligodendrocyte precursor cells; O4, oligodendrocyte marker O4; MBP, myelin basic protein.

    Techniques Used: Isolation, Immunostaining, Staining, Derivative Assay, Marker

    8) Product Images from "Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody"

    Article Title: Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14793-z

    3D8 scFv colocalizes intracellularly with proteoglycans and caveolin-1 upon binding to cell surface proteoglycans (HSPGs and CSPGs). ( a,b ) Confocal microscopy to detect intracellular colocalization of 3D8 scFv, proteoglycans (HSPGs and CSPGs), and caveolin-1. HeLa cells were incubated with 3D8 scFv (10 μM) for 1 h, 6 h, or 12 h at 37 °C. After fixation and permeabilization, cells were incubated at 4 °C overnight with a primary antibody mixture comprising rabbit (IgG) anti-3D8 scFv, mouse (IgG) anti-caveolin-1, and mouse (IgM) anti-HS antibodies ( a ), or a mixture of rabbit anti-3D8 scFv, mouse anti-caveolin-1, and mouse (IgM) anti-CS antibodies ( b ). Thereafter, cells were incubated with a mixture of Alexa Fluor 647-conjugated goat anti-rabbit IgG, TRITC-conjugated goat anti-mouse IgG, and Alexa Fluor 488-conjugated goat anti-mouse IgM/μ chain-specific antibody. Enlarged images of the boxed areas in the upper panel are shown in the lower panel. ( c,d ) HeLa cells were incubated for 6 h at 37 °C with HW6, followed rabbit anti-His tag and Alexa Fluor 647-conjugated goat anti-rabbit IgG antibodies. Bar , 10 μm.
    Figure Legend Snippet: 3D8 scFv colocalizes intracellularly with proteoglycans and caveolin-1 upon binding to cell surface proteoglycans (HSPGs and CSPGs). ( a,b ) Confocal microscopy to detect intracellular colocalization of 3D8 scFv, proteoglycans (HSPGs and CSPGs), and caveolin-1. HeLa cells were incubated with 3D8 scFv (10 μM) for 1 h, 6 h, or 12 h at 37 °C. After fixation and permeabilization, cells were incubated at 4 °C overnight with a primary antibody mixture comprising rabbit (IgG) anti-3D8 scFv, mouse (IgG) anti-caveolin-1, and mouse (IgM) anti-HS antibodies ( a ), or a mixture of rabbit anti-3D8 scFv, mouse anti-caveolin-1, and mouse (IgM) anti-CS antibodies ( b ). Thereafter, cells were incubated with a mixture of Alexa Fluor 647-conjugated goat anti-rabbit IgG, TRITC-conjugated goat anti-mouse IgG, and Alexa Fluor 488-conjugated goat anti-mouse IgM/μ chain-specific antibody. Enlarged images of the boxed areas in the upper panel are shown in the lower panel. ( c,d ) HeLa cells were incubated for 6 h at 37 °C with HW6, followed rabbit anti-His tag and Alexa Fluor 647-conjugated goat anti-rabbit IgG antibodies. Bar , 10 μm.

    Techniques Used: Binding Assay, Confocal Microscopy, Incubation

    3D8 scFv binds to cell surface HSPGs and CSPGs. ( a ) Flow cytometry analysis of cell surface expression of endogenous HSPGs and CSPGs in HeLa cells. ( b ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs and CSPGs. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) and HW6 scFv (10 μM) (negative control). Thereafter, cells were incubated with a primary antibody mixture containing a rabbit anti-3D8 scFv antibody and a mouse IgM anti-HS antibody, or a rabbit anti-3D8 scFv antibody and a mouse IgM anti-CS antibody. After washing, cells were incubated with a secondary antibody mixture comprising TRITC-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgM. ( c ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs in the presence of soluble HS and CS chains. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) in the absence ( upper panel ) or presence ( middle or lower panel ) of heparin (10 μg/ml) or CS-A (10 μg/ml), followed by the procedures described in ( b ). ( d,e ) Flow cytometry ( d ) and confocal microscopy ( e ) to detect the cell surface sugar chains (HS and CS) and cell surface binding of 3D8 scFv to HeLa cells pre-treated with heparinase III (10 mIU/ml) and chondroitinase ABC (100 mIU/ml). ( b,c,e ) Nuclei were stained with Hoechst 33342 (blue). Bar , 10 μm.
    Figure Legend Snippet: 3D8 scFv binds to cell surface HSPGs and CSPGs. ( a ) Flow cytometry analysis of cell surface expression of endogenous HSPGs and CSPGs in HeLa cells. ( b ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs and CSPGs. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) and HW6 scFv (10 μM) (negative control). Thereafter, cells were incubated with a primary antibody mixture containing a rabbit anti-3D8 scFv antibody and a mouse IgM anti-HS antibody, or a rabbit anti-3D8 scFv antibody and a mouse IgM anti-CS antibody. After washing, cells were incubated with a secondary antibody mixture comprising TRITC-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgM. ( c ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs in the presence of soluble HS and CS chains. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) in the absence ( upper panel ) or presence ( middle or lower panel ) of heparin (10 μg/ml) or CS-A (10 μg/ml), followed by the procedures described in ( b ). ( d,e ) Flow cytometry ( d ) and confocal microscopy ( e ) to detect the cell surface sugar chains (HS and CS) and cell surface binding of 3D8 scFv to HeLa cells pre-treated with heparinase III (10 mIU/ml) and chondroitinase ABC (100 mIU/ml). ( b,c,e ) Nuclei were stained with Hoechst 33342 (blue). Bar , 10 μm.

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Confocal Microscopy, Binding Assay, Incubation, Negative Control, Staining

    9) Product Images from "Toll-Like Receptor 9 Activation Rescues Impaired Antibody Response in Needle-free Intradermal DNA Vaccination"

    Article Title: Toll-Like Receptor 9 Activation Rescues Impaired Antibody Response in Needle-free Intradermal DNA Vaccination

    Journal: Scientific Reports

    doi: 10.1038/srep33564

    Suboptimal IgG, but not the IgM, antibody response following the NF-ID immunization. Groups of mice, C57BL/6 or BALB/c, were immunized with the pCMV-Gag DNA vector through the NF-ID, or needle-syringe IM or ID route. Each symbol represents an individual animal. The sera obtained 11 days following the booster immunization were diluted 100 and 500-fold for the measurement of IgG in C57BL/6 and BALB/c mice respectively. The IgM from both the strains of mice was measured in serum diluted 100-fold. The assay employed secondary antibodies conjugated to HRP specific to IgM or IgG isotypes. The mean absorbance value of the group and the SEM are shown. The dotted line represents the cut-off value defined as two times the mean absorbance value of the control group of four animals immunized with the null vector. The data of the NF-ID and IM groups were obtained from three independent experiments containing 4–6 mice per group. The data of the needle-syringe ID group were obtained from two independent experiments containing 4 or 6 animals per group. The one-way ANOVA was used for statistical comparison (****p
    Figure Legend Snippet: Suboptimal IgG, but not the IgM, antibody response following the NF-ID immunization. Groups of mice, C57BL/6 or BALB/c, were immunized with the pCMV-Gag DNA vector through the NF-ID, or needle-syringe IM or ID route. Each symbol represents an individual animal. The sera obtained 11 days following the booster immunization were diluted 100 and 500-fold for the measurement of IgG in C57BL/6 and BALB/c mice respectively. The IgM from both the strains of mice was measured in serum diluted 100-fold. The assay employed secondary antibodies conjugated to HRP specific to IgM or IgG isotypes. The mean absorbance value of the group and the SEM are shown. The dotted line represents the cut-off value defined as two times the mean absorbance value of the control group of four animals immunized with the null vector. The data of the NF-ID and IM groups were obtained from three independent experiments containing 4–6 mice per group. The data of the needle-syringe ID group were obtained from two independent experiments containing 4 or 6 animals per group. The one-way ANOVA was used for statistical comparison (****p

    Techniques Used: Mouse Assay, Plasmid Preparation

    10) Product Images from "A Glucuronoxylomannan Epitope Exhibits Serotype-Specific Accessibility and Redistributes towards the Capsule Surface during Titanization of the Fungal Pathogen Cryptococcus neoformans"

    Article Title: A Glucuronoxylomannan Epitope Exhibits Serotype-Specific Accessibility and Redistributes towards the Capsule Surface during Titanization of the Fungal Pathogen Cryptococcus neoformans

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00731-18

    The Crp127 epitope is spatially confined to distinct capsular regions, and binding elicits capsular swelling reactions distinct from those elicited by 18B7.  Cryptococcus  cells were grown under capsule-inducing conditions and imaged to determine the location of the Crp127 epitope within the enlarged capsule and characterize the capsular reaction patterns elicited by this antibody. (A to D)  Cryptococcus  strains of serotype A (KN99α and H99) (A), serotype B (R265 and CDCR272) (B), serotype D (B3501 and JEC155) (C), and serotype AD (CBS 950 and ZG287) (D) were labeled for chitin (CFW) (blue) and Crp127 (goat Alexa 647-conjugated anti-mouse IgM μ-chain) (far red), suspended in India ink to visualize the capsule, and imaged using confocal microscopy. Representative images of a single focal plane are shown for each strain. Presented are images merged for transmitted light and Crp127 (left panels) and Crp127 and chitin (right panels). (E to H) Capsule-induced cells of strains KN99α (E), R265 (F), B3501 (G), and CBS 950 (H) were also left untreated (top right panels) or treated with mAb 18B7 (top right panels) or with mAb Crp127 (bottom panels) and imaged using DIC microscopy to observe capsular reaction patterns. Bars, 5 μm.
    Figure Legend Snippet: The Crp127 epitope is spatially confined to distinct capsular regions, and binding elicits capsular swelling reactions distinct from those elicited by 18B7. Cryptococcus cells were grown under capsule-inducing conditions and imaged to determine the location of the Crp127 epitope within the enlarged capsule and characterize the capsular reaction patterns elicited by this antibody. (A to D) Cryptococcus strains of serotype A (KN99α and H99) (A), serotype B (R265 and CDCR272) (B), serotype D (B3501 and JEC155) (C), and serotype AD (CBS 950 and ZG287) (D) were labeled for chitin (CFW) (blue) and Crp127 (goat Alexa 647-conjugated anti-mouse IgM μ-chain) (far red), suspended in India ink to visualize the capsule, and imaged using confocal microscopy. Representative images of a single focal plane are shown for each strain. Presented are images merged for transmitted light and Crp127 (left panels) and Crp127 and chitin (right panels). (E to H) Capsule-induced cells of strains KN99α (E), R265 (F), B3501 (G), and CBS 950 (H) were also left untreated (top right panels) or treated with mAb 18B7 (top right panels) or with mAb Crp127 (bottom panels) and imaged using DIC microscopy to observe capsular reaction patterns. Bars, 5 μm.

    Techniques Used: Binding Assay, Labeling, Confocal Microscopy, Microscopy

    11) Product Images from "Comparative adaptations in oxidative and glycolytic muscle fibers in a low voluntary wheel running rat model performing three levels of physical activity"

    Article Title: Comparative adaptations in oxidative and glycolytic muscle fibers in a low voluntary wheel running rat model performing three levels of physical activity

    Journal: Physiological Reports

    doi: 10.14814/phy2.12619

    Myosin heavy chain isoform expression in soleus and plantaris. Representative images are shown to the right-hand side of the graphs. Fiber membranes are highlighted by dystrophin stain (red). Fiber types can be identified as MHC I (blue), MHC IIa (green), and all remaining fibers were classified as MCH IIb/x (black). *Different from SEDENTARY. n = 6–7/group.
    Figure Legend Snippet: Myosin heavy chain isoform expression in soleus and plantaris. Representative images are shown to the right-hand side of the graphs. Fiber membranes are highlighted by dystrophin stain (red). Fiber types can be identified as MHC I (blue), MHC IIa (green), and all remaining fibers were classified as MCH IIb/x (black). *Different from SEDENTARY. n = 6–7/group.

    Techniques Used: Expressing, Staining

    12) Product Images from "Protection against Candidiasis by an Immunoglobulin G3 (IgG3) Monoclonal Antibody Specific for the Same Mannotriose as an IgM Protective Antibody"

    Article Title: Protection against Candidiasis by an Immunoglobulin G3 (IgG3) Monoclonal Antibody Specific for the Same Mannotriose as an IgM Protective Antibody

    Journal: Infection and Immunity

    doi:

    The C3.1 epitope is uniformly distributed on the cell surface of yeast forms of C. albicans . Hydrophilic stationary-phase yeast cells were reacted with MAb C3.1, washed, and counterreacted with fluorescence-labeled anti-mouse IgG. The C3.1 epitope is located over the entire cell surface (A). The same field was photographed under phase-contrast microscopy (B). The C3.1 epitope distribution displays the same homogeneous pattern over the cell surface as the epitope recognized by the IgM protective antibody, MAb IgM B6.1. These results provide further evidence that the IgG3 antibody is specific for the same oligomannoside as the protective IgM. Note that some yeast cells (arrows) and all of the blastoconidia (arrowheads) were nonreactive with MAb C3.1. The importance of nonreactive cells in pathogenesis and protection experiments is unknown. Bar, 10 μM.
    Figure Legend Snippet: The C3.1 epitope is uniformly distributed on the cell surface of yeast forms of C. albicans . Hydrophilic stationary-phase yeast cells were reacted with MAb C3.1, washed, and counterreacted with fluorescence-labeled anti-mouse IgG. The C3.1 epitope is located over the entire cell surface (A). The same field was photographed under phase-contrast microscopy (B). The C3.1 epitope distribution displays the same homogeneous pattern over the cell surface as the epitope recognized by the IgM protective antibody, MAb IgM B6.1. These results provide further evidence that the IgG3 antibody is specific for the same oligomannoside as the protective IgM. Note that some yeast cells (arrows) and all of the blastoconidia (arrowheads) were nonreactive with MAb C3.1. The importance of nonreactive cells in pathogenesis and protection experiments is unknown. Bar, 10 μM.

    Techniques Used: Fluorescence, Labeling, Microscopy

    13) Product Images from "Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance"

    Article Title: Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038313

    HL 2°Abs exhibit background and detection bias independent of 1° and 2°Ab concentrations. (A) FLISAs showing detection of different concentrations of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs as indicated by the values on the X-axes, with HL 2°Ab (top row), and respective SCS 2°Abs (middle row), at the concentrations indicated above the columns. Bottom row shows data from the graphs in the top row normalized to values for the IgG1 mAb. (B) HL bias is seen at all 2°Ab concentrations tested in transiently transfected COS-1 cells. Immunofluorescence labeling of Kv1.2-expressing COS-1 cells, probed with 5 µg/mL of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs and different amounts of HL 2°Ab (red), and the respective SCS 2°Abs (green), as indicated on the X-axis. The Y-axis is the red∶green (HL∶SCS) fluorescence ratio (in arbitrary units). (C) Immunoblots showing lack of crossreactivity in SCS 2°Ab detection of antigens loaded at great excess. Recombinant GST fusion proteins containing different amounts of Kv1.2 and PSD95 antigens, and GST alone, were size fractionated on a single SDS gel and transferred to an immunoblot. Amounts loaded of GST-PSD95 ranged from 4–972 ng, as indicated below lower left panel, and for GST-Kv1.2 and GST alone from 972–4 ng, as indicated below lower right panel. The immunoblot was simultaneously probed with anti-Kv1.2 K14/16 (IgG2b, red), anti-PSD95 K28/43 (IgG2a, blue) and anti-GST N100/13 (IgG1, green), and corresponding SCS 2°Abs. Lane to left of top left panel shows molecular weight standards in kDa. Image reveals a lack of crossreactivity between SCS 2°Abs and bound 1°Abs even under conditions of excess antigen.
    Figure Legend Snippet: HL 2°Abs exhibit background and detection bias independent of 1° and 2°Ab concentrations. (A) FLISAs showing detection of different concentrations of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs as indicated by the values on the X-axes, with HL 2°Ab (top row), and respective SCS 2°Abs (middle row), at the concentrations indicated above the columns. Bottom row shows data from the graphs in the top row normalized to values for the IgG1 mAb. (B) HL bias is seen at all 2°Ab concentrations tested in transiently transfected COS-1 cells. Immunofluorescence labeling of Kv1.2-expressing COS-1 cells, probed with 5 µg/mL of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs and different amounts of HL 2°Ab (red), and the respective SCS 2°Abs (green), as indicated on the X-axis. The Y-axis is the red∶green (HL∶SCS) fluorescence ratio (in arbitrary units). (C) Immunoblots showing lack of crossreactivity in SCS 2°Ab detection of antigens loaded at great excess. Recombinant GST fusion proteins containing different amounts of Kv1.2 and PSD95 antigens, and GST alone, were size fractionated on a single SDS gel and transferred to an immunoblot. Amounts loaded of GST-PSD95 ranged from 4–972 ng, as indicated below lower left panel, and for GST-Kv1.2 and GST alone from 972–4 ng, as indicated below lower right panel. The immunoblot was simultaneously probed with anti-Kv1.2 K14/16 (IgG2b, red), anti-PSD95 K28/43 (IgG2a, blue) and anti-GST N100/13 (IgG1, green), and corresponding SCS 2°Abs. Lane to left of top left panel shows molecular weight standards in kDa. Image reveals a lack of crossreactivity between SCS 2°Abs and bound 1°Abs even under conditions of excess antigen.

    Techniques Used: Transfection, Immunofluorescence, Labeling, Expressing, Fluorescence, Western Blot, Recombinant, SDS-Gel, Molecular Weight

    HL 2°Abs show a bias for mAbs of different IgG subclasses in a variety of applications. (A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid as labeled, probed with anti-PSD-95 (IgG2a), anti-Kv1.2 (IgG2b) and anti-Kv2.1 (IgG1) mAbs, and HL 2°Ab (green), and a cocktail (1∶1∶1) of SCS anti-IgG1, -IgG2a and -IgG2b 2°Abs (red). Multicolor panel is original immunoblot; single color panels are images of separated colors. Changes in tint reflect bias of HL for (more green) IgG2a > IgG2b > IgG1 (more red). Lane to left shows molecular weight standards in kDa. (B) FLISAs show that IgG subclass bias of HL is present at all concentrations of 1°Abs. Left panel: SCS 2°Abs (each at 1 µg/ml). Right panel: HL 2°Ab. Circles: L76/36 IgG2a; triangles; K14/16 IgG2b; squares: K14/39 IgG1. (C) IgG subclass bias is also present in immunofluorescence labeling of Kv1.2-expressing COS-1 cells. Cells were labeled with mAb as noted, and HL 2°Ab (red), and SCS 2°Abs (green) as detailed in Methods . Changes in red∶green tint reflect bias of HL for (more red) IgG2a > IgG2b > IgG1 (more green). Scale bar = 100 µm. Panel to right is quantitation of immunocytochemistry results from three fields each of three independent samples.
    Figure Legend Snippet: HL 2°Abs show a bias for mAbs of different IgG subclasses in a variety of applications. (A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid as labeled, probed with anti-PSD-95 (IgG2a), anti-Kv1.2 (IgG2b) and anti-Kv2.1 (IgG1) mAbs, and HL 2°Ab (green), and a cocktail (1∶1∶1) of SCS anti-IgG1, -IgG2a and -IgG2b 2°Abs (red). Multicolor panel is original immunoblot; single color panels are images of separated colors. Changes in tint reflect bias of HL for (more green) IgG2a > IgG2b > IgG1 (more red). Lane to left shows molecular weight standards in kDa. (B) FLISAs show that IgG subclass bias of HL is present at all concentrations of 1°Abs. Left panel: SCS 2°Abs (each at 1 µg/ml). Right panel: HL 2°Ab. Circles: L76/36 IgG2a; triangles; K14/16 IgG2b; squares: K14/39 IgG1. (C) IgG subclass bias is also present in immunofluorescence labeling of Kv1.2-expressing COS-1 cells. Cells were labeled with mAb as noted, and HL 2°Ab (red), and SCS 2°Abs (green) as detailed in Methods . Changes in red∶green tint reflect bias of HL for (more red) IgG2a > IgG2b > IgG1 (more green). Scale bar = 100 µm. Panel to right is quantitation of immunocytochemistry results from three fields each of three independent samples.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Labeling, Molecular Weight, Immunofluorescence, Quantitation Assay, Immunocytochemistry

    HL detection bias is seen in 2°Ab preparations from different suppliers, with different fluorophores, and with enzyme conjugates. (A) Kv1.2-transfected COS-1 cells were labeled with 1°Ab as in Figure 4C , and HL 2°Ab and the respective SCS 2°Abs, and the ratios of fluorescence intensities from three fields each of three independent samples normalized to the HL/IgG1 ratio. Letters are supplier (L = Life Technologies, R = Rockland), numbers are Alex or DyLight fluorophore conjugates; high: highly adsorbed; fab: F(ab′) 2 fragment of HL ( e.g. , L488 SCS is Life Technologies Alexa 488 conjugated SCS). 4/09 and 7/11 refer to two lots of Life Technologies HL. (B) FLISAs showing detection bias of 2°Abs is present at all 2°Ab concentrations. Upper left: Life Technologies HL. Upper right: Life Technologies SCS. Lower left: Jackson ImmunoResearch HL. Lower right: Jackson ImmunoResearch HL (highly cross-adsorbed). (C) HRP conjugated HL secondaries show detection bias by immunoblot. Purified mAb IgG preparations were analyzed by reducing SDS-PAGE and coomassie blue staining (CB), or immunoblotting and detection with two different HRP-conjugated H+L 2°Abs and ECL. HL: Kirkegaard Perry Laboratories. HL*: Antibodies Incorporated. Note subclass-specific differences in detection of heavy chain (HC) but not light chain (LC) bands in IgG preparations.
    Figure Legend Snippet: HL detection bias is seen in 2°Ab preparations from different suppliers, with different fluorophores, and with enzyme conjugates. (A) Kv1.2-transfected COS-1 cells were labeled with 1°Ab as in Figure 4C , and HL 2°Ab and the respective SCS 2°Abs, and the ratios of fluorescence intensities from three fields each of three independent samples normalized to the HL/IgG1 ratio. Letters are supplier (L = Life Technologies, R = Rockland), numbers are Alex or DyLight fluorophore conjugates; high: highly adsorbed; fab: F(ab′) 2 fragment of HL ( e.g. , L488 SCS is Life Technologies Alexa 488 conjugated SCS). 4/09 and 7/11 refer to two lots of Life Technologies HL. (B) FLISAs showing detection bias of 2°Abs is present at all 2°Ab concentrations. Upper left: Life Technologies HL. Upper right: Life Technologies SCS. Lower left: Jackson ImmunoResearch HL. Lower right: Jackson ImmunoResearch HL (highly cross-adsorbed). (C) HRP conjugated HL secondaries show detection bias by immunoblot. Purified mAb IgG preparations were analyzed by reducing SDS-PAGE and coomassie blue staining (CB), or immunoblotting and detection with two different HRP-conjugated H+L 2°Abs and ECL. HL: Kirkegaard Perry Laboratories. HL*: Antibodies Incorporated. Note subclass-specific differences in detection of heavy chain (HC) but not light chain (LC) bands in IgG preparations.

    Techniques Used: Transfection, Labeling, Fluorescence, Purification, SDS Page, Staining

    Analysis of knockout mouse tissue reveals increased background of HL 2°Abs. Sections of brains from WT and Kv2.1 knockout (KO) mice were labeled with an anti-Kv2.1 IgG1 mAb, or in vehicle alone (bottom row, no 1°Ab), followed by simultaneous detection with both HL (green) and IgG1-specific (red) 2°Abs. Columns represent samples with different [2°Ab] as in column header. All samples were imaged using identical exposure times. Note that the panels in the top row are the same as those in the WT row but showing the green channel only. Scale bar = 25 µm.
    Figure Legend Snippet: Analysis of knockout mouse tissue reveals increased background of HL 2°Abs. Sections of brains from WT and Kv2.1 knockout (KO) mice were labeled with an anti-Kv2.1 IgG1 mAb, or in vehicle alone (bottom row, no 1°Ab), followed by simultaneous detection with both HL (green) and IgG1-specific (red) 2°Abs. Columns represent samples with different [2°Ab] as in column header. All samples were imaged using identical exposure times. Note that the panels in the top row are the same as those in the WT row but showing the green channel only. Scale bar = 25 µm.

    Techniques Used: Knock-Out, Mouse Assay, Labeling

    14) Product Images from "MOG cell-based assay detects non-MS patients with inflammatory neurologic disease"

    Article Title: MOG cell-based assay detects non-MS patients with inflammatory neurologic disease

    Journal: Neurology® Neuroimmunology & Neuroinflammation

    doi: 10.1212/NXI.0000000000000089

    Antibodies to MOG using different secondary antibodies: Anti-human IgG (H + L), IgG1, or IgM (A) Comparison of binding to full-length myelin oligodendrocyte glycoprotein (FL-MOG) using anti-human IgG (H + L), anti-IgM, or anti-IgG1 secondary antibodies with 3 different test sera (a-c) and a healthy control serum (con). (B) IgM and (C) IgG1 binding scores for patients and healthy controls (HC). (D.a) PIRES2-DsRed2-FL-MOG transiently transfected HEK cells are separated into cells that express MOG and DsRed2 well (in the upper section of the graph) or poorly or not at all (lowest section of the graph). (D.b) Healthy control sera (upper panels) causes a specific shift in the MOG-transfected cells compared to the untransfected cells when anti-human IgG (H + L) or anti-human IgM secondary antibodies are used (arrows), but not when anti-human IgG1 secondary antibodies are used. The lower panels show higher shifts in sera positive for FL-MOG antibodies compared to controls in the upper panel. (E) Fifteen samples that were IgG (H + L) positive and 5 healthy controls were tested on flow cytometry with anti-IgM or IgG1. A high cutoff is generated with anti-human IgM secondary antibody (ΔMFI of 270) vs a ΔMFI of 2.5 for the anti-human IgG1 antibody. Of note, one IgM-positive patient is IgG1 negative (blue circle). Ab = antibody; AQP4 = aquaporin-4; CBA = cell-based assay; MFI = mean fluorescence intensity; MS = multiple sclerosis.
    Figure Legend Snippet: Antibodies to MOG using different secondary antibodies: Anti-human IgG (H + L), IgG1, or IgM (A) Comparison of binding to full-length myelin oligodendrocyte glycoprotein (FL-MOG) using anti-human IgG (H + L), anti-IgM, or anti-IgG1 secondary antibodies with 3 different test sera (a-c) and a healthy control serum (con). (B) IgM and (C) IgG1 binding scores for patients and healthy controls (HC). (D.a) PIRES2-DsRed2-FL-MOG transiently transfected HEK cells are separated into cells that express MOG and DsRed2 well (in the upper section of the graph) or poorly or not at all (lowest section of the graph). (D.b) Healthy control sera (upper panels) causes a specific shift in the MOG-transfected cells compared to the untransfected cells when anti-human IgG (H + L) or anti-human IgM secondary antibodies are used (arrows), but not when anti-human IgG1 secondary antibodies are used. The lower panels show higher shifts in sera positive for FL-MOG antibodies compared to controls in the upper panel. (E) Fifteen samples that were IgG (H + L) positive and 5 healthy controls were tested on flow cytometry with anti-IgM or IgG1. A high cutoff is generated with anti-human IgM secondary antibody (ΔMFI of 270) vs a ΔMFI of 2.5 for the anti-human IgG1 antibody. Of note, one IgM-positive patient is IgG1 negative (blue circle). Ab = antibody; AQP4 = aquaporin-4; CBA = cell-based assay; MFI = mean fluorescence intensity; MS = multiple sclerosis.

    Techniques Used: Binding Assay, Transfection, Flow Cytometry, Cytometry, Generated, Crocin Bleaching Assay, Cell Based Assay, Fluorescence, Mass Spectrometry

    15) Product Images from "Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody"

    Article Title: Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14793-z

    3D8 scFv colocalizes intracellularly with proteoglycans and caveolin-1 upon binding to cell surface proteoglycans (HSPGs and CSPGs). ( a,b ) Confocal microscopy to detect intracellular colocalization of 3D8 scFv, proteoglycans (HSPGs and CSPGs), and caveolin-1. HeLa cells were incubated with 3D8 scFv (10 μM) for 1 h, 6 h, or 12 h at 37 °C. After fixation and permeabilization, cells were incubated at 4 °C overnight with a primary antibody mixture comprising rabbit (IgG) anti-3D8 scFv, mouse (IgG) anti-caveolin-1, and mouse (IgM) anti-HS antibodies ( a ), or a mixture of rabbit anti-3D8 scFv, mouse anti-caveolin-1, and mouse (IgM) anti-CS antibodies ( b ). Thereafter, cells were incubated with a mixture of Alexa Fluor 647-conjugated goat anti-rabbit IgG, TRITC-conjugated goat anti-mouse IgG, and Alexa Fluor 488-conjugated goat anti-mouse IgM/μ chain-specific antibody. Enlarged images of the boxed areas in the upper panel are shown in the lower panel. ( c,d ) HeLa cells were incubated for 6 h at 37 °C with HW6, followed rabbit anti-His tag and Alexa Fluor 647-conjugated goat anti-rabbit IgG antibodies. Bar , 10 μm.
    Figure Legend Snippet: 3D8 scFv colocalizes intracellularly with proteoglycans and caveolin-1 upon binding to cell surface proteoglycans (HSPGs and CSPGs). ( a,b ) Confocal microscopy to detect intracellular colocalization of 3D8 scFv, proteoglycans (HSPGs and CSPGs), and caveolin-1. HeLa cells were incubated with 3D8 scFv (10 μM) for 1 h, 6 h, or 12 h at 37 °C. After fixation and permeabilization, cells were incubated at 4 °C overnight with a primary antibody mixture comprising rabbit (IgG) anti-3D8 scFv, mouse (IgG) anti-caveolin-1, and mouse (IgM) anti-HS antibodies ( a ), or a mixture of rabbit anti-3D8 scFv, mouse anti-caveolin-1, and mouse (IgM) anti-CS antibodies ( b ). Thereafter, cells were incubated with a mixture of Alexa Fluor 647-conjugated goat anti-rabbit IgG, TRITC-conjugated goat anti-mouse IgG, and Alexa Fluor 488-conjugated goat anti-mouse IgM/μ chain-specific antibody. Enlarged images of the boxed areas in the upper panel are shown in the lower panel. ( c,d ) HeLa cells were incubated for 6 h at 37 °C with HW6, followed rabbit anti-His tag and Alexa Fluor 647-conjugated goat anti-rabbit IgG antibodies. Bar , 10 μm.

    Techniques Used: Binding Assay, Confocal Microscopy, Incubation

    3D8 scFv binds to cell surface HSPGs and CSPGs. ( a ) Flow cytometry analysis of cell surface expression of endogenous HSPGs and CSPGs in HeLa cells. ( b ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs and CSPGs. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) and HW6 scFv (10 μM) (negative control). Thereafter, cells were incubated with a primary antibody mixture containing a rabbit anti-3D8 scFv antibody and a mouse IgM anti-HS antibody, or a rabbit anti-3D8 scFv antibody and a mouse IgM anti-CS antibody. After washing, cells were incubated with a secondary antibody mixture comprising TRITC-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgM. ( c ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs in the presence of soluble HS and CS chains. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) in the absence ( upper panel ) or presence ( middle or lower panel ) of heparin (10 μg/ml) or CS-A (10 μg/ml), followed by the procedures described in ( b ). ( d,e ) Flow cytometry ( d ) and confocal microscopy ( e ) to detect the cell surface sugar chains (HS and CS) and cell surface binding of 3D8 scFv to HeLa cells pre-treated with heparinase III (10 mIU/ml) and chondroitinase ABC (100 mIU/ml). ( b,c,e ) Nuclei were stained with Hoechst 33342 (blue). Bar , 10 μm.
    Figure Legend Snippet: 3D8 scFv binds to cell surface HSPGs and CSPGs. ( a ) Flow cytometry analysis of cell surface expression of endogenous HSPGs and CSPGs in HeLa cells. ( b ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs and CSPGs. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) and HW6 scFv (10 μM) (negative control). Thereafter, cells were incubated with a primary antibody mixture containing a rabbit anti-3D8 scFv antibody and a mouse IgM anti-HS antibody, or a rabbit anti-3D8 scFv antibody and a mouse IgM anti-CS antibody. After washing, cells were incubated with a secondary antibody mixture comprising TRITC-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgM. ( c ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs in the presence of soluble HS and CS chains. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) in the absence ( upper panel ) or presence ( middle or lower panel ) of heparin (10 μg/ml) or CS-A (10 μg/ml), followed by the procedures described in ( b ). ( d,e ) Flow cytometry ( d ) and confocal microscopy ( e ) to detect the cell surface sugar chains (HS and CS) and cell surface binding of 3D8 scFv to HeLa cells pre-treated with heparinase III (10 mIU/ml) and chondroitinase ABC (100 mIU/ml). ( b,c,e ) Nuclei were stained with Hoechst 33342 (blue). Bar , 10 μm.

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Confocal Microscopy, Binding Assay, Incubation, Negative Control, Staining

    16) Product Images from "CXCL12 induces migration of oligodendrocyte precursor cells through the CXCR4-activated MEK/ERK and PI3K/AKT pathways"

    Article Title: CXCL12 induces migration of oligodendrocyte precursor cells through the CXCR4-activated MEK/ERK and PI3K/AKT pathways

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2018.9444

    Isolation and identification of OPCs. (A) Immunostaining demonstrated that OPCs were positive for NG2. (B) Immunostaining demonstrated that OPCs were positive for PDGFR-α. (C) Immunostaining showed that the differentiated OPCs were positive for O4. (D) Immunostaining showed that the differentiated OPCs were positive for MBP. Nuclei were stained with DAPI (blue). NG2, neural/glial antigen 2; PDGFR-α, platelet-derived growth factor receptor-α; OPCs, oligodendrocyte precursor cells; O4, oligodendrocyte marker O4; MBP, myelin basic protein.
    Figure Legend Snippet: Isolation and identification of OPCs. (A) Immunostaining demonstrated that OPCs were positive for NG2. (B) Immunostaining demonstrated that OPCs were positive for PDGFR-α. (C) Immunostaining showed that the differentiated OPCs were positive for O4. (D) Immunostaining showed that the differentiated OPCs were positive for MBP. Nuclei were stained with DAPI (blue). NG2, neural/glial antigen 2; PDGFR-α, platelet-derived growth factor receptor-α; OPCs, oligodendrocyte precursor cells; O4, oligodendrocyte marker O4; MBP, myelin basic protein.

    Techniques Used: Isolation, Immunostaining, Staining, Derivative Assay, Marker

    17) Product Images from "4-AMINOPYRIDINE ATTENUATES MUSCLE ATROPHY AFTER SCIATIC NERVE CRUSH INJURY IN MICE"

    Article Title: 4-AMINOPYRIDINE ATTENUATES MUSCLE ATROPHY AFTER SCIATIC NERVE CRUSH INJURY IN MICE

    Journal: Muscle & nerve

    doi: 10.1002/mus.26516

    In vivo  4-AP treatment increases Pax7- and Ki67-expressing SCs in TA and EDL muscles of injured limb.  (A)  Representative Pax7/Ki67/DAPI IF images in CTL muscles at day 0; each image represents 9 images from 3 different mice.  (B)  Representative images of Pax7 (red), Ki67 (green), and DAPI (blue) in 4-AP-treated group at day 14 postinjury; each image represents 9 images from 3 different mice.  (C)  Representative Pax7/Ki67/DAPI IF images in Sal and 4-AP-treated groups at days 7 and 14 postinjury. Frozen muscle cross-sections were incubated simultaneously with 2 different primary antibodies (Pax7 mouse antibody and Ki67 rabbit antibody) and 2 different secondary antibodies (AlexaFluor 594-conjugated goat anti-mouse IgG and AlexaFluor 488-conjugated goat anti-rabbit IgM). In addition to individual red (Pax7) or green (Ki67) color, combination of red and green colors resulted in yellow cells; each image represents 9 images from 3 different mice.  (D,E)  The pooled number of Pax7 +  ( D ) and Ki67 +  ( E ) cells in uninjured and injured groups with or without treatment;  n  = 3/group. * P
    Figure Legend Snippet: In vivo 4-AP treatment increases Pax7- and Ki67-expressing SCs in TA and EDL muscles of injured limb. (A) Representative Pax7/Ki67/DAPI IF images in CTL muscles at day 0; each image represents 9 images from 3 different mice. (B) Representative images of Pax7 (red), Ki67 (green), and DAPI (blue) in 4-AP-treated group at day 14 postinjury; each image represents 9 images from 3 different mice. (C) Representative Pax7/Ki67/DAPI IF images in Sal and 4-AP-treated groups at days 7 and 14 postinjury. Frozen muscle cross-sections were incubated simultaneously with 2 different primary antibodies (Pax7 mouse antibody and Ki67 rabbit antibody) and 2 different secondary antibodies (AlexaFluor 594-conjugated goat anti-mouse IgG and AlexaFluor 488-conjugated goat anti-rabbit IgM). In addition to individual red (Pax7) or green (Ki67) color, combination of red and green colors resulted in yellow cells; each image represents 9 images from 3 different mice. (D,E) The pooled number of Pax7 + ( D ) and Ki67 + ( E ) cells in uninjured and injured groups with or without treatment; n = 3/group. * P

    Techniques Used: In Vivo, Expressing, Mouse Assay, Incubation

    18) Product Images from "Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance"

    Article Title: Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038313

    HL 2°Abs show a bias for immunohistochemistry labeling with mAbs of different IgG subclasses. Rat brain sections were labeled with the same concentrations of a single mAb, and a rabbit anti-Kv2.1 pAb, followed by detection with SCS (left column) or HL (right column) 2°Abs, (red), and anti-rabbit IgG (green), each at 1 µg/ml. Top row: anti-Kv4.2 IgG1; middle row: anti-BK channel IgG2a; and bottom row: anti-Kv1.2 IgG2b. Each row was imaged at the same exposure times. Scale bar = 50 µm for panels in top two rows, and 25 µm for panels in bottom row.
    Figure Legend Snippet: HL 2°Abs show a bias for immunohistochemistry labeling with mAbs of different IgG subclasses. Rat brain sections were labeled with the same concentrations of a single mAb, and a rabbit anti-Kv2.1 pAb, followed by detection with SCS (left column) or HL (right column) 2°Abs, (red), and anti-rabbit IgG (green), each at 1 µg/ml. Top row: anti-Kv4.2 IgG1; middle row: anti-BK channel IgG2a; and bottom row: anti-Kv1.2 IgG2b. Each row was imaged at the same exposure times. Scale bar = 50 µm for panels in top two rows, and 25 µm for panels in bottom row.

    Techniques Used: Immunohistochemistry, Labeling

    HL 2°Abs exhibit background and detection bias independent of 1° and 2°Ab concentrations. (A) FLISAs showing detection of different concentrations of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs as indicated by the values on the X-axes, with HL 2°Ab (top row), and respective SCS 2°Abs (middle row), at the concentrations indicated above the columns. Bottom row shows data from the graphs in the top row normalized to values for the IgG1 mAb. (B) HL bias is seen at all 2°Ab concentrations tested in transiently transfected COS-1 cells. Immunofluorescence labeling of Kv1.2-expressing COS-1 cells, probed with 5 µg/mL of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs and different amounts of HL 2°Ab (red), and the respective SCS 2°Abs (green), as indicated on the X-axis. The Y-axis is the red∶green (HL∶SCS) fluorescence ratio (in arbitrary units). (C) Immunoblots showing lack of crossreactivity in SCS 2°Ab detection of antigens loaded at great excess. Recombinant GST fusion proteins containing different amounts of Kv1.2 and PSD95 antigens, and GST alone, were size fractionated on a single SDS gel and transferred to an immunoblot. Amounts loaded of GST-PSD95 ranged from 4–972 ng, as indicated below lower left panel, and for GST-Kv1.2 and GST alone from 972–4 ng, as indicated below lower right panel. The immunoblot was simultaneously probed with anti-Kv1.2 K14/16 (IgG2b, red), anti-PSD95 K28/43 (IgG2a, blue) and anti-GST N100/13 (IgG1, green), and corresponding SCS 2°Abs. Lane to left of top left panel shows molecular weight standards in kDa. Image reveals a lack of crossreactivity between SCS 2°Abs and bound 1°Abs even under conditions of excess antigen.
    Figure Legend Snippet: HL 2°Abs exhibit background and detection bias independent of 1° and 2°Ab concentrations. (A) FLISAs showing detection of different concentrations of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs as indicated by the values on the X-axes, with HL 2°Ab (top row), and respective SCS 2°Abs (middle row), at the concentrations indicated above the columns. Bottom row shows data from the graphs in the top row normalized to values for the IgG1 mAb. (B) HL bias is seen at all 2°Ab concentrations tested in transiently transfected COS-1 cells. Immunofluorescence labeling of Kv1.2-expressing COS-1 cells, probed with 5 µg/mL of IgG1 (K14/39, squares), IgG2a (L76/36, circles), and IgG2b (K14/16, triangles) mAbs and different amounts of HL 2°Ab (red), and the respective SCS 2°Abs (green), as indicated on the X-axis. The Y-axis is the red∶green (HL∶SCS) fluorescence ratio (in arbitrary units). (C) Immunoblots showing lack of crossreactivity in SCS 2°Ab detection of antigens loaded at great excess. Recombinant GST fusion proteins containing different amounts of Kv1.2 and PSD95 antigens, and GST alone, were size fractionated on a single SDS gel and transferred to an immunoblot. Amounts loaded of GST-PSD95 ranged from 4–972 ng, as indicated below lower left panel, and for GST-Kv1.2 and GST alone from 972–4 ng, as indicated below lower right panel. The immunoblot was simultaneously probed with anti-Kv1.2 K14/16 (IgG2b, red), anti-PSD95 K28/43 (IgG2a, blue) and anti-GST N100/13 (IgG1, green), and corresponding SCS 2°Abs. Lane to left of top left panel shows molecular weight standards in kDa. Image reveals a lack of crossreactivity between SCS 2°Abs and bound 1°Abs even under conditions of excess antigen.

    Techniques Used: Transfection, Immunofluorescence, Labeling, Expressing, Fluorescence, Western Blot, Recombinant, SDS-Gel, Molecular Weight

    HL 2°Abs show a bias for mAbs of different IgG subclasses in a variety of applications. (A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid as labeled, probed with anti-PSD-95 (IgG2a), anti-Kv1.2 (IgG2b) and anti-Kv2.1 (IgG1) mAbs, and HL 2°Ab (green), and a cocktail (1∶1∶1) of SCS anti-IgG1, -IgG2a and -IgG2b 2°Abs (red). Multicolor panel is original immunoblot; single color panels are images of separated colors. Changes in tint reflect bias of HL for (more green) IgG2a > IgG2b > IgG1 (more red). Lane to left shows molecular weight standards in kDa. (B) FLISAs show that IgG subclass bias of HL is present at all concentrations of 1°Abs. Left panel: SCS 2°Abs (each at 1 µg/ml). Right panel: HL 2°Ab. Circles: L76/36 IgG2a; triangles; K14/16 IgG2b; squares: K14/39 IgG1. (C) IgG subclass bias is also present in immunofluorescence labeling of Kv1.2-expressing COS-1 cells. Cells were labeled with mAb as noted, and HL 2°Ab (red), and SCS 2°Abs (green) as detailed in Methods . Changes in red∶green tint reflect bias of HL for (more red) IgG2a > IgG2b > IgG1 (more green). Scale bar = 100 µm. Panel to right is quantitation of immunocytochemistry results from three fields each of three independent samples.
    Figure Legend Snippet: HL 2°Abs show a bias for mAbs of different IgG subclasses in a variety of applications. (A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid as labeled, probed with anti-PSD-95 (IgG2a), anti-Kv1.2 (IgG2b) and anti-Kv2.1 (IgG1) mAbs, and HL 2°Ab (green), and a cocktail (1∶1∶1) of SCS anti-IgG1, -IgG2a and -IgG2b 2°Abs (red). Multicolor panel is original immunoblot; single color panels are images of separated colors. Changes in tint reflect bias of HL for (more green) IgG2a > IgG2b > IgG1 (more red). Lane to left shows molecular weight standards in kDa. (B) FLISAs show that IgG subclass bias of HL is present at all concentrations of 1°Abs. Left panel: SCS 2°Abs (each at 1 µg/ml). Right panel: HL 2°Ab. Circles: L76/36 IgG2a; triangles; K14/16 IgG2b; squares: K14/39 IgG1. (C) IgG subclass bias is also present in immunofluorescence labeling of Kv1.2-expressing COS-1 cells. Cells were labeled with mAb as noted, and HL 2°Ab (red), and SCS 2°Abs (green) as detailed in Methods . Changes in red∶green tint reflect bias of HL for (more red) IgG2a > IgG2b > IgG1 (more green). Scale bar = 100 µm. Panel to right is quantitation of immunocytochemistry results from three fields each of three independent samples.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Labeling, Molecular Weight, Immunofluorescence, Quantitation Assay, Immunocytochemistry

    HL detection bias is seen in 2°Ab preparations from different suppliers, with different fluorophores, and with enzyme conjugates. (A) Kv1.2-transfected COS-1 cells were labeled with 1°Ab as in Figure 4C , and HL 2°Ab and the respective SCS 2°Abs, and the ratios of fluorescence intensities from three fields each of three independent samples normalized to the HL/IgG1 ratio. Letters are supplier (L = Life Technologies, R = Rockland), numbers are Alex or DyLight fluorophore conjugates; high: highly adsorbed; fab: F(ab′) 2 fragment of HL ( e.g. , L488 SCS is Life Technologies Alexa 488 conjugated SCS). 4/09 and 7/11 refer to two lots of Life Technologies HL. (B) FLISAs showing detection bias of 2°Abs is present at all 2°Ab concentrations. Upper left: Life Technologies HL. Upper right: Life Technologies SCS. Lower left: Jackson ImmunoResearch HL. Lower right: Jackson ImmunoResearch HL (highly cross-adsorbed). (C) HRP conjugated HL secondaries show detection bias by immunoblot. Purified mAb IgG preparations were analyzed by reducing SDS-PAGE and coomassie blue staining (CB), or immunoblotting and detection with two different HRP-conjugated H+L 2°Abs and ECL. HL: Kirkegaard Perry Laboratories. HL*: Antibodies Incorporated. Note subclass-specific differences in detection of heavy chain (HC) but not light chain (LC) bands in IgG preparations.
    Figure Legend Snippet: HL detection bias is seen in 2°Ab preparations from different suppliers, with different fluorophores, and with enzyme conjugates. (A) Kv1.2-transfected COS-1 cells were labeled with 1°Ab as in Figure 4C , and HL 2°Ab and the respective SCS 2°Abs, and the ratios of fluorescence intensities from three fields each of three independent samples normalized to the HL/IgG1 ratio. Letters are supplier (L = Life Technologies, R = Rockland), numbers are Alex or DyLight fluorophore conjugates; high: highly adsorbed; fab: F(ab′) 2 fragment of HL ( e.g. , L488 SCS is Life Technologies Alexa 488 conjugated SCS). 4/09 and 7/11 refer to two lots of Life Technologies HL. (B) FLISAs showing detection bias of 2°Abs is present at all 2°Ab concentrations. Upper left: Life Technologies HL. Upper right: Life Technologies SCS. Lower left: Jackson ImmunoResearch HL. Lower right: Jackson ImmunoResearch HL (highly cross-adsorbed). (C) HRP conjugated HL secondaries show detection bias by immunoblot. Purified mAb IgG preparations were analyzed by reducing SDS-PAGE and coomassie blue staining (CB), or immunoblotting and detection with two different HRP-conjugated H+L 2°Abs and ECL. HL: Kirkegaard Perry Laboratories. HL*: Antibodies Incorporated. Note subclass-specific differences in detection of heavy chain (HC) but not light chain (LC) bands in IgG preparations.

    Techniques Used: Transfection, Labeling, Fluorescence, Purification, SDS Page, Staining

    Analysis of knockout mouse tissue reveals increased background of HL 2°Abs. Sections of brains from WT and Kv2.1 knockout (KO) mice were labeled with an anti-Kv2.1 IgG1 mAb, or in vehicle alone (bottom row, no 1°Ab), followed by simultaneous detection with both HL (green) and IgG1-specific (red) 2°Abs. Columns represent samples with different [2°Ab] as in column header. All samples were imaged using identical exposure times. Note that the panels in the top row are the same as those in the WT row but showing the green channel only. Scale bar = 25 µm.
    Figure Legend Snippet: Analysis of knockout mouse tissue reveals increased background of HL 2°Abs. Sections of brains from WT and Kv2.1 knockout (KO) mice were labeled with an anti-Kv2.1 IgG1 mAb, or in vehicle alone (bottom row, no 1°Ab), followed by simultaneous detection with both HL (green) and IgG1-specific (red) 2°Abs. Columns represent samples with different [2°Ab] as in column header. All samples were imaged using identical exposure times. Note that the panels in the top row are the same as those in the WT row but showing the green channel only. Scale bar = 25 µm.

    Techniques Used: Knock-Out, Mouse Assay, Labeling

    SCS 2°Abs yield robust and reliable simultaneous triple labeling with three mAbs on immunoblots and in rat brain sections. (A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid, probed with anti-PSD-95 (IgG2a, blue), anti-Kv1.2 (IgG2b, red) and anti-Kv2.1 (IgG1, green), and SCS 2°Abs. Multicolor panel is original immunoblot; single color panels are images of separated colors. Lane to left shows molecular weight standards in kDa. Note differential post-translational modification of target proteins in brain versus heterologous cells alters their relative electrophoretic mobility. B–E. Images show specific and non-overlapping labeling for (B) Kv4.2 (green), (C) QKI (red), (D) and BK channels (blue), and (E) merge of all three, in a rat brain section, showing the region containing the entire cerebellum. Inset in E shows boxed area of cerebellar cortex. Labels mark the molecular layer (ML), Purkinje cell layer (PCL), and granule cell layer (GCL). Scale bar on Panel E = 500 µm.
    Figure Legend Snippet: SCS 2°Abs yield robust and reliable simultaneous triple labeling with three mAbs on immunoblots and in rat brain sections. (A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid, probed with anti-PSD-95 (IgG2a, blue), anti-Kv1.2 (IgG2b, red) and anti-Kv2.1 (IgG1, green), and SCS 2°Abs. Multicolor panel is original immunoblot; single color panels are images of separated colors. Lane to left shows molecular weight standards in kDa. Note differential post-translational modification of target proteins in brain versus heterologous cells alters their relative electrophoretic mobility. B–E. Images show specific and non-overlapping labeling for (B) Kv4.2 (green), (C) QKI (red), (D) and BK channels (blue), and (E) merge of all three, in a rat brain section, showing the region containing the entire cerebellum. Inset in E shows boxed area of cerebellar cortex. Labels mark the molecular layer (ML), Purkinje cell layer (PCL), and granule cell layer (GCL). Scale bar on Panel E = 500 µm.

    Techniques Used: Labeling, Western Blot, Transfection, Expressing, Plasmid Preparation, Molecular Weight, Modification

    19) Product Images from "EAF2 mediates germinal centre B-cell apoptosis to suppress excessive immune responses and prevent autoimmunity"

    Article Title: EAF2 mediates germinal centre B-cell apoptosis to suppress excessive immune responses and prevent autoimmunity

    Journal: Nature Communications

    doi: 10.1038/ncomms10836

    Increased NP-specific Ab in immunized Eaf2 −/− mice. ( a ) Five pairs of WT and Eaf2 −/− mice were immunized with 10 μg of NP–CGG in alum. Serum levels of NP-specific IgG 1 Ab were measured with NP25-BSA (upper) and NP2-BSA (lower) at the indicated time points as described in Methods section. ( b ) AFC in spleen (left) and BM (right) at 6 weeks after NP–CGG immunization. NP2 and NP25 detect high affinity and total NP-specific AFC, respectively. ( c ) NP-specific IgM (upper) and IgG 3 (lower) serum Ab levels after immunization with NP-Ficoll. The results of five pairs of WT and Eaf2 −/− mice are shown. ( d ) NP-specific IgM AFC in the spleen and BM at 21 weeks after NP-Ficoll immunization. Three pairs of WT and Eaf2 −/− mice were analysed. ( e ) and ( f ), Rag1 −/− mice reconstituted with WT T+WT B or WT T+ Eaf2 −/− B cells were immunized with NP–CGG and analysed for ( e ) NP-specific total IgG 1 Ab (NP25) and high-affinity IgG 1 Ab (NP2) in the serum and ( f ) AFC in the spleen. Open circles, Rag1 −/− mice reconstituted with WT T+WT B cells; Solid circles, Rag1 −/− mice reconstituted with WT T+ Eaf2 −/− B cells. Arrows in a and c indicate immunization times. The red bar indicates the mean value of each group. * P
    Figure Legend Snippet: Increased NP-specific Ab in immunized Eaf2 −/− mice. ( a ) Five pairs of WT and Eaf2 −/− mice were immunized with 10 μg of NP–CGG in alum. Serum levels of NP-specific IgG 1 Ab were measured with NP25-BSA (upper) and NP2-BSA (lower) at the indicated time points as described in Methods section. ( b ) AFC in spleen (left) and BM (right) at 6 weeks after NP–CGG immunization. NP2 and NP25 detect high affinity and total NP-specific AFC, respectively. ( c ) NP-specific IgM (upper) and IgG 3 (lower) serum Ab levels after immunization with NP-Ficoll. The results of five pairs of WT and Eaf2 −/− mice are shown. ( d ) NP-specific IgM AFC in the spleen and BM at 21 weeks after NP-Ficoll immunization. Three pairs of WT and Eaf2 −/− mice were analysed. ( e ) and ( f ), Rag1 −/− mice reconstituted with WT T+WT B or WT T+ Eaf2 −/− B cells were immunized with NP–CGG and analysed for ( e ) NP-specific total IgG 1 Ab (NP25) and high-affinity IgG 1 Ab (NP2) in the serum and ( f ) AFC in the spleen. Open circles, Rag1 −/− mice reconstituted with WT T+WT B cells; Solid circles, Rag1 −/− mice reconstituted with WT T+ Eaf2 −/− B cells. Arrows in a and c indicate immunization times. The red bar indicates the mean value of each group. * P

    Techniques Used: Mouse Assay

    20) Product Images from "Homeostatic expansion of autoreactive immunoglobulin-secreting cells in the Rag2 mouse model of Omenn syndrome"

    Article Title: Homeostatic expansion of autoreactive immunoglobulin-secreting cells in the Rag2 mouse model of Omenn syndrome

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20091928

    Residual autoreactive B cells in Rag2 R229Q mice contribute to tissue damage in autoimmune-like manifestations. (A) Characterization of kidney infiltrates and correlation with clinical symptoms. Kidney sections from a mouse with proteinuria (score 4: ≥ 2000 mg/dl; top) and a mouse without renal disease (bottom). Kidneys from mice with proteinuria show infiltrates positive for staining with CD3, B220, IgM and IgG. Bars: (left) 100 µM; (middle) 50 µM; (right) 60 µM. (B) The presence of IgG anti–double-strand DNA autoantibodies was analyzed in the sera of WT ( n = 70) and Rag2 R229Q ( n = 97) mice by ELISA and IFA in five independent experiments. Bars indicate the frequency of positive sera in mice aged 8–24 wk. (C) Ig targeting tissues are detected in the sera of Rag2 R229Q mice. Frozen sections obtained from, gut, thyroid, and adrenal gland of Rag2/Il2rc double-KO mice were incubated with sera obtained from WT and Rag2 R229Q mice. Staining with anti–mouse IgG Ab followed by incubation with secondary, anti–mouse IgG peroxidase-conjugated antibody was performed. Representative images from 1 out of 10 mice tested/group. Bars, 100 µM. (D) FACS analysis of λ light chain expression in gated B220 + IgM + cells. Results are representative of 11 mice/group analyzed in three independent experiments. (E) Serum BAFF in Rag2 R229Q ( n = 26) and WT littermates ( n = 15), measured by ELISA in two independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001.
    Figure Legend Snippet: Residual autoreactive B cells in Rag2 R229Q mice contribute to tissue damage in autoimmune-like manifestations. (A) Characterization of kidney infiltrates and correlation with clinical symptoms. Kidney sections from a mouse with proteinuria (score 4: ≥ 2000 mg/dl; top) and a mouse without renal disease (bottom). Kidneys from mice with proteinuria show infiltrates positive for staining with CD3, B220, IgM and IgG. Bars: (left) 100 µM; (middle) 50 µM; (right) 60 µM. (B) The presence of IgG anti–double-strand DNA autoantibodies was analyzed in the sera of WT ( n = 70) and Rag2 R229Q ( n = 97) mice by ELISA and IFA in five independent experiments. Bars indicate the frequency of positive sera in mice aged 8–24 wk. (C) Ig targeting tissues are detected in the sera of Rag2 R229Q mice. Frozen sections obtained from, gut, thyroid, and adrenal gland of Rag2/Il2rc double-KO mice were incubated with sera obtained from WT and Rag2 R229Q mice. Staining with anti–mouse IgG Ab followed by incubation with secondary, anti–mouse IgG peroxidase-conjugated antibody was performed. Representative images from 1 out of 10 mice tested/group. Bars, 100 µM. (D) FACS analysis of λ light chain expression in gated B220 + IgM + cells. Results are representative of 11 mice/group analyzed in three independent experiments. (E) Serum BAFF in Rag2 R229Q ( n = 26) and WT littermates ( n = 15), measured by ELISA in two independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001.

    Techniques Used: Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Incubation, FACS, Expressing

    Characterization of the B cell compartment in Rag2 R229Q mice. (A) Analysis of circulating B cells from WT and Rag2 R229Q mice. (B) Total BM cells were labeled with anti-B220, CD43, IgM, and IgD to determine B cell developmental stages. Representative FACS plots are shown for mice (WT = 35 and Rag2 R229Q = 42) analyzed in six independent experiments. Numbers indicate percentage of cells for each gate. (C) Dot plots show analysis of splenic mature and transitional B cell populations from WT and Rag2 R229Q mice, using IgD and IgM markers. Histograms show expression of CD40, CD86, CD69, and MHCII activation markers in gated splenic B220 + IgM + cells. Numbers indicate percentage of cells for each gate. (D) Splenocytes were labeled with anti-B220, -CD21, and -CD23 and analyzed by flow cytometry. Dot plots are gated on B220 + cells. Numbers indicate percentage of cells for each gate. The results shown are representative of mice (WT = 43 and Rag2 R229Q = 57) analyzed in six independent experiments.
    Figure Legend Snippet: Characterization of the B cell compartment in Rag2 R229Q mice. (A) Analysis of circulating B cells from WT and Rag2 R229Q mice. (B) Total BM cells were labeled with anti-B220, CD43, IgM, and IgD to determine B cell developmental stages. Representative FACS plots are shown for mice (WT = 35 and Rag2 R229Q = 42) analyzed in six independent experiments. Numbers indicate percentage of cells for each gate. (C) Dot plots show analysis of splenic mature and transitional B cell populations from WT and Rag2 R229Q mice, using IgD and IgM markers. Histograms show expression of CD40, CD86, CD69, and MHCII activation markers in gated splenic B220 + IgM + cells. Numbers indicate percentage of cells for each gate. (D) Splenocytes were labeled with anti-B220, -CD21, and -CD23 and analyzed by flow cytometry. Dot plots are gated on B220 + cells. Numbers indicate percentage of cells for each gate. The results shown are representative of mice (WT = 43 and Rag2 R229Q = 57) analyzed in six independent experiments.

    Techniques Used: Mouse Assay, Labeling, FACS, Expressing, Activation Assay, Flow Cytometry, Cytometry

    Rag2 R229Q mice have impaired proliferative responses to TLR agonists but intact Ig responses. (A) Splenic B220 + lymphocytes were purified by cell sorting, and the relative mRNA levels of TLR4 and TLR9 expression were determined by RT-PCR. The samples were run in duplicates. The obtained values were normalized to 18S rRNA and indicated as arbitrary units (A.U.). Mean values ± SD of three independent experiments testing eight mice/group. (B) Total splenocytes were CFSE labeled, and then stimulated in vitro with LPS (10 µg/ml) and CpG (2.5 µg/ml) in the absence or presence of IL-4 (10 ng/ml). CFSE dilution was analyzed by FACS at day 4 of culture. Representative FACS histograms of CFSE profile in B220 + cell gate are shown (percentage is reported above the gate). The histogram plot is representative of three independent experiments (C) In parallel, Ig levels were measured in the culture supernatants. Ig concentrations were normalized for the number of B220 + cells in the culture. *, P
    Figure Legend Snippet: Rag2 R229Q mice have impaired proliferative responses to TLR agonists but intact Ig responses. (A) Splenic B220 + lymphocytes were purified by cell sorting, and the relative mRNA levels of TLR4 and TLR9 expression were determined by RT-PCR. The samples were run in duplicates. The obtained values were normalized to 18S rRNA and indicated as arbitrary units (A.U.). Mean values ± SD of three independent experiments testing eight mice/group. (B) Total splenocytes were CFSE labeled, and then stimulated in vitro with LPS (10 µg/ml) and CpG (2.5 µg/ml) in the absence or presence of IL-4 (10 ng/ml). CFSE dilution was analyzed by FACS at day 4 of culture. Representative FACS histograms of CFSE profile in B220 + cell gate are shown (percentage is reported above the gate). The histogram plot is representative of three independent experiments (C) In parallel, Ig levels were measured in the culture supernatants. Ig concentrations were normalized for the number of B220 + cells in the culture. *, P

    Techniques Used: Mouse Assay, Purification, FACS, Expressing, Reverse Transcription Polymerase Chain Reaction, Labeling, In Vitro

    CD4 T cells sustain ISC differentiation in Rag2 R229Q mice. Rag2 R229Q mice (5 wk-old) were injected i.v. with two doses of anti-CD4 mAb (0.25 mg/mouse) 3 wk apart and were analyzed after 7 wk. (A) Representative FACS plots of CD4 and CD8 stainings in treated mice and controls. (B) ISC by ELISpot assay. Data represent the mean ± SD number of spots obtained from n = 5–6 mice/group analyzed and normalized to 10 5 B220 + cells. Serum Ig (C) and cytokine (D) levels in treated and control mice. Data represent the mean ± SD of concentrations observed in n = 5–6 mice/group analyzed. *, P
    Figure Legend Snippet: CD4 T cells sustain ISC differentiation in Rag2 R229Q mice. Rag2 R229Q mice (5 wk-old) were injected i.v. with two doses of anti-CD4 mAb (0.25 mg/mouse) 3 wk apart and were analyzed after 7 wk. (A) Representative FACS plots of CD4 and CD8 stainings in treated mice and controls. (B) ISC by ELISpot assay. Data represent the mean ± SD number of spots obtained from n = 5–6 mice/group analyzed and normalized to 10 5 B220 + cells. Serum Ig (C) and cytokine (D) levels in treated and control mice. Data represent the mean ± SD of concentrations observed in n = 5–6 mice/group analyzed. *, P

    Techniques Used: Mouse Assay, Injection, FACS, Enzyme-linked Immunospot

    Serum Ig levels and ISC in lymphoid organs. (A) Sera from naive WT ( n = 49) and Rag2 R229Q ( n = 67) mice (5–37 wk-old) were collected and their IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA concentrations were determined by luminex technique using a Bioplex reader. Each serum sample was run in duplicate, and five independent assays were performed. Serum IgE concentrations were measured by ELISA in five independent experiments. (B) The frequency of IgM-, IgG-, IgE-, and IgA-secreting cells in the BM, spleen, and LNs of naive WT and Rag2 R229Q mice (10–12 wk old) was determined by ELISpot. The number of spots/10 5 B220 + cells is indicated. Results are mean ± SD of five independent experiments testing a total of 17 mice/group. (C) Transcriptional programming sustains ISC differentiation in Rag2 R229Q mice. B220 + lymphocytes were purified by cell sorter from total splenocytes of WT ( n = 6) and Rag2 R229Q mice ( n = 12). Expression levels of different transcription factors were normalized to 18S rRNA. Quantitative RT-PCR was run in triplicates. Relative measures are indicated as arbitrary units (A.U.). Data represent one of the three independent experiments with consistent results. *, P
    Figure Legend Snippet: Serum Ig levels and ISC in lymphoid organs. (A) Sera from naive WT ( n = 49) and Rag2 R229Q ( n = 67) mice (5–37 wk-old) were collected and their IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA concentrations were determined by luminex technique using a Bioplex reader. Each serum sample was run in duplicate, and five independent assays were performed. Serum IgE concentrations were measured by ELISA in five independent experiments. (B) The frequency of IgM-, IgG-, IgE-, and IgA-secreting cells in the BM, spleen, and LNs of naive WT and Rag2 R229Q mice (10–12 wk old) was determined by ELISpot. The number of spots/10 5 B220 + cells is indicated. Results are mean ± SD of five independent experiments testing a total of 17 mice/group. (C) Transcriptional programming sustains ISC differentiation in Rag2 R229Q mice. B220 + lymphocytes were purified by cell sorter from total splenocytes of WT ( n = 6) and Rag2 R229Q mice ( n = 12). Expression levels of different transcription factors were normalized to 18S rRNA. Quantitative RT-PCR was run in triplicates. Relative measures are indicated as arbitrary units (A.U.). Data represent one of the three independent experiments with consistent results. *, P

    Techniques Used: Mouse Assay, Luminex, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Purification, Expressing, Quantitative RT-PCR

    Effect of anti-BAFF-R mAb administration in Rag2 R229Q mice. Mutant mice were treated with three doses of 0.5 mg anti–BAFF-R mAb or PBS, and followed for a total period of 2 mo. (A) Splenic B220 + cell counts. (B) Absolute numbers of splenic ISC. Mean values ± SD obtained from n = 6–7 mice/group. (C) The global and cell-specific (CD3 + , B220 + , IgM + ) degree of infiltration was scored in different organs (liver, kidney, lung, and gut) from anti–BAFF-R mAb and PBS-treated mice. Bars indicate the mean ± SD infiltration index calculated for all the organs from n = 6–7 mice/group. Images show immunostains of gut and liver from representative PBS and anti–BAFF-R mAb-treated mice. Bars: (left) 200 µM; (right) 50 µM. (D) The presence of high-affinity IgG anti-dsDNA autoantibodies was analyzed in the sera of PBS and anti–BAFF-R Rag2 R229Q mice by IFA, before and after the treatment. Bars indicate the frequency of positive sera. Proteinuria was assessed before and after treatment of Rag2 R229Q mice with PBS and anti–BAFF-R, respectively. Mean values ± SD of proteinuria index in n = 6–7 mice/group. (bottom) Representative images of glomeruli from untreated proteinuric mice and anti-BAFFR–treated mice. Bars, 50 µM.
    Figure Legend Snippet: Effect of anti-BAFF-R mAb administration in Rag2 R229Q mice. Mutant mice were treated with three doses of 0.5 mg anti–BAFF-R mAb or PBS, and followed for a total period of 2 mo. (A) Splenic B220 + cell counts. (B) Absolute numbers of splenic ISC. Mean values ± SD obtained from n = 6–7 mice/group. (C) The global and cell-specific (CD3 + , B220 + , IgM + ) degree of infiltration was scored in different organs (liver, kidney, lung, and gut) from anti–BAFF-R mAb and PBS-treated mice. Bars indicate the mean ± SD infiltration index calculated for all the organs from n = 6–7 mice/group. Images show immunostains of gut and liver from representative PBS and anti–BAFF-R mAb-treated mice. Bars: (left) 200 µM; (right) 50 µM. (D) The presence of high-affinity IgG anti-dsDNA autoantibodies was analyzed in the sera of PBS and anti–BAFF-R Rag2 R229Q mice by IFA, before and after the treatment. Bars indicate the frequency of positive sera. Proteinuria was assessed before and after treatment of Rag2 R229Q mice with PBS and anti–BAFF-R, respectively. Mean values ± SD of proteinuria index in n = 6–7 mice/group. (bottom) Representative images of glomeruli from untreated proteinuric mice and anti-BAFFR–treated mice. Bars, 50 µM.

    Techniques Used: Mouse Assay, Mutagenesis, Immunofluorescence

    Rag2 R229Q mice fail to induce specific humoral response to OVA. WT and Rag2 R229Q mice were immunized into the footpad with OVA emulsified in CFA and boosted at day 21 in IFA. PBS was injected in control mice. Total IgG1 (A) and anti-OVA–specific IgG (B) antibody titers were determined at days 26, 33, and 48 after challenge by ELISA. Each point represents mean values + SD from n = 3–4 mice and were corrected for background binding. Frequency analysis of total (C) and specific (D) ISCs by ELISpot assay on splenocytes from WT and Rag2 R229Q mice at the indicated times after OVA challenge. Data represent the mean ± SD number of spots obtained from n = 3–4 mice/group analyzed in two independent experiments and normalized to 10 5 B220 + cells. n.d., not detectable. CTL, PBS-treated control mice. *, P
    Figure Legend Snippet: Rag2 R229Q mice fail to induce specific humoral response to OVA. WT and Rag2 R229Q mice were immunized into the footpad with OVA emulsified in CFA and boosted at day 21 in IFA. PBS was injected in control mice. Total IgG1 (A) and anti-OVA–specific IgG (B) antibody titers were determined at days 26, 33, and 48 after challenge by ELISA. Each point represents mean values + SD from n = 3–4 mice and were corrected for background binding. Frequency analysis of total (C) and specific (D) ISCs by ELISpot assay on splenocytes from WT and Rag2 R229Q mice at the indicated times after OVA challenge. Data represent the mean ± SD number of spots obtained from n = 3–4 mice/group analyzed in two independent experiments and normalized to 10 5 B220 + cells. n.d., not detectable. CTL, PBS-treated control mice. *, P

    Techniques Used: Mouse Assay, Immunofluorescence, Injection, Enzyme-linked Immunosorbent Assay, Binding Assay, Enzyme-linked Immunospot, CTL Assay

    21) Product Images from "The Ox40/Ox40 Ligand Pathway Promotes Pathogenic Th Cell Responses, Plasmablast Accumulation, and Lupus Nephritis in NZB/W F1 Mice"

    Article Title: The Ox40/Ox40 Ligand Pathway Promotes Pathogenic Th Cell Responses, Plasmablast Accumulation, and Lupus Nephritis in NZB/W F1 Mice

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1700608

    Anti-Ox40 agonist mAb treatment exacerbates renal disease and IgM deposition in NZB/W F1 mice. Renal disease monitoring following anti-Ox40 agonist mAb (red) and a control Ig (blue) treatment (10 mg/kg, three times per week for 3 wk) in NZB/W F1 mice. Black arrows (Rx) refer to treatment days. The age and proteinuria status of each cohort (at the time of first treatment) is indicated at the top of each plot. ( A ) Thirteen-week-old proteinuria-free NZB/W F1 mice at treatment start. ( B ) Three independent cohorts (21-, 26-, and 27-wk-old) of NZB/W F1 mice with proteinuria 30–100 mg/dl at treatment start. ( C ) Representative H E- and PAS-stained kidney sections (left and middle panels) highlighting glomeruli (top panels), periarterial regions (middle panels), and tubulointerstitium (bottom panels). Scale bars, 100 μm. Corresponding disease severity scores (right panels). Group means are plotted in black ( n = 36 per group combined). ( D ) Representative images from frozen kidney sections (left panels) stained for glomerular deposits of IgM (top panel), IgG (middle panel), and C3 (bottom panel). Scale bars, 50 μm. Corresponding signal intensity in the cortex regions is presented as pixel intensity/μM 2 of cortex with group mean ± SD from two independent cohorts ( n = 25–28 per group, combined) (right panels). * p
    Figure Legend Snippet: Anti-Ox40 agonist mAb treatment exacerbates renal disease and IgM deposition in NZB/W F1 mice. Renal disease monitoring following anti-Ox40 agonist mAb (red) and a control Ig (blue) treatment (10 mg/kg, three times per week for 3 wk) in NZB/W F1 mice. Black arrows (Rx) refer to treatment days. The age and proteinuria status of each cohort (at the time of first treatment) is indicated at the top of each plot. ( A ) Thirteen-week-old proteinuria-free NZB/W F1 mice at treatment start. ( B ) Three independent cohorts (21-, 26-, and 27-wk-old) of NZB/W F1 mice with proteinuria 30–100 mg/dl at treatment start. ( C ) Representative H E- and PAS-stained kidney sections (left and middle panels) highlighting glomeruli (top panels), periarterial regions (middle panels), and tubulointerstitium (bottom panels). Scale bars, 100 μm. Corresponding disease severity scores (right panels). Group means are plotted in black ( n = 36 per group combined). ( D ) Representative images from frozen kidney sections (left panels) stained for glomerular deposits of IgM (top panel), IgG (middle panel), and C3 (bottom panel). Scale bars, 50 μm. Corresponding signal intensity in the cortex regions is presented as pixel intensity/μM 2 of cortex with group mean ± SD from two independent cohorts ( n = 25–28 per group, combined) (right panels). * p

    Techniques Used: Mouse Assay, Staining

    Ox40-stimulated splenic T cell response, plasmablast accumulation, and Ig effects. Quantification of total serum IgM concentration ( A ) and total serum IgG1, IgG2a, IgG2b, and IgG3 concentrations ( B ) following anti-Ox40 mAb treatment presented as mean ± SD combined from three independent cohorts ( n = 24 for IgG1 and 36 for all other groups, combined). ( C – E ) Representative flow cytometry plots (left panels) and summary data (right panels) from NZB/W F1 spleen preparations presented with mean ± SD. (C) Frequency of activation/memory cell markers CD44 and CD62L on Tconv cells (gated live CD45 + CD3 + CD4 + Foxp3 − ). (D) Frequency and total number of CXCR5 + ICOS + Tfh cells (gated CD3 + CD4 + ). (E) Frequency and total number of plasmablast cells (CD138 + ). ( F ) Total number of IgM + CD138 + plasmablast cells. * p
    Figure Legend Snippet: Ox40-stimulated splenic T cell response, plasmablast accumulation, and Ig effects. Quantification of total serum IgM concentration ( A ) and total serum IgG1, IgG2a, IgG2b, and IgG3 concentrations ( B ) following anti-Ox40 mAb treatment presented as mean ± SD combined from three independent cohorts ( n = 24 for IgG1 and 36 for all other groups, combined). ( C – E ) Representative flow cytometry plots (left panels) and summary data (right panels) from NZB/W F1 spleen preparations presented with mean ± SD. (C) Frequency of activation/memory cell markers CD44 and CD62L on Tconv cells (gated live CD45 + CD3 + CD4 + Foxp3 − ). (D) Frequency and total number of CXCR5 + ICOS + Tfh cells (gated CD3 + CD4 + ). (E) Frequency and total number of plasmablast cells (CD138 + ). ( F ) Total number of IgM + CD138 + plasmablast cells. * p

    Techniques Used: Concentration Assay, Flow Cytometry, Cytometry, Activation Assay

    22) Product Images from "Hspa13 Promotes Plasma Cell Production and Antibody Secretion"

    Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.00913

    Hspa13 cKO reduced sheep red blood cell (SRC)-induced PB/PC and antibody production. Nine-week-old female Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were injected intraperitoneally (i.p.) with 1 × 10 9 SRCs on days 0 and 7. (A,B) Hspa13 cKO did not affect SRC-induce GC B-cell production. On day 21 following SRC stimulation, splenic lymphocytes were stained with isotype control antibodies, anti-mouse CD19, B220, CD38, and GL7 antibodies, and were then analyzed by FACS. The percentages (A) and the absolute numbers (B) of CD38 lo GL7 hi GC cells gated on CD19 + B220 + are shown. (C,D) Hspa13 cKO did not affect the SRC-induced dark zone (DZ) and light zone (LZ) GC B-cell production. On day 21 following SRC stimulation, splenic lymphocytes were stained with anti-mouse CD19, B220, CD38, GL7, CXCR4, and CD86 antibodies, and were then analyzed by FACS. The percentages (C) and the absolute numbers (D) of CXCR4 hi CD86 lo DZ and CXCR4 lo CD86 hi LZ GC B cells gated on CD19 + B220 + CD38 lo GL7 hi GC cells are shown. (E,F) Hspa13 cKO reduced SRC-induced IgG1-, IgG2b-, IgG2c-, and IgG3-expressing PBs/PCs. On day 21 following SRC stimulation, splenic lymphocytes were collected and intracellular staining was performed with isotype control antibodies, anti-mouse B220, IgG1, IgG2b, IgG2c, and IgG3 antibodies. The percentages (E) and the absolute numbers (F) of IgG1-, IgG2b-, IgG2c-, and IgG3-expressing B220 + PBs and B220 − PCs are shown. (G) Hspa13 cKO reduced SRC-induced antibody secretion. On day 21 following SRC stimulation, sera were collected and the total IgM, IgG, IgG1, IgG2b, IgG2c, and IgG3 antibody levels were analyzed by ELISA. (A–G) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by two-way (D) and one-way (B,F,G) ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P
    Figure Legend Snippet: Hspa13 cKO reduced sheep red blood cell (SRC)-induced PB/PC and antibody production. Nine-week-old female Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were injected intraperitoneally (i.p.) with 1 × 10 9 SRCs on days 0 and 7. (A,B) Hspa13 cKO did not affect SRC-induce GC B-cell production. On day 21 following SRC stimulation, splenic lymphocytes were stained with isotype control antibodies, anti-mouse CD19, B220, CD38, and GL7 antibodies, and were then analyzed by FACS. The percentages (A) and the absolute numbers (B) of CD38 lo GL7 hi GC cells gated on CD19 + B220 + are shown. (C,D) Hspa13 cKO did not affect the SRC-induced dark zone (DZ) and light zone (LZ) GC B-cell production. On day 21 following SRC stimulation, splenic lymphocytes were stained with anti-mouse CD19, B220, CD38, GL7, CXCR4, and CD86 antibodies, and were then analyzed by FACS. The percentages (C) and the absolute numbers (D) of CXCR4 hi CD86 lo DZ and CXCR4 lo CD86 hi LZ GC B cells gated on CD19 + B220 + CD38 lo GL7 hi GC cells are shown. (E,F) Hspa13 cKO reduced SRC-induced IgG1-, IgG2b-, IgG2c-, and IgG3-expressing PBs/PCs. On day 21 following SRC stimulation, splenic lymphocytes were collected and intracellular staining was performed with isotype control antibodies, anti-mouse B220, IgG1, IgG2b, IgG2c, and IgG3 antibodies. The percentages (E) and the absolute numbers (F) of IgG1-, IgG2b-, IgG2c-, and IgG3-expressing B220 + PBs and B220 − PCs are shown. (G) Hspa13 cKO reduced SRC-induced antibody secretion. On day 21 following SRC stimulation, sera were collected and the total IgM, IgG, IgG1, IgG2b, IgG2c, and IgG3 antibody levels were analyzed by ELISA. (A–G) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by two-way (D) and one-way (B,F,G) ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P

    Techniques Used: Mouse Assay, Injection, Staining, FACS, Expressing, Enzyme-linked Immunosorbent Assay

    Hspa13 cKO reduced class switch recombination (CSR), somatic hypermutation (SHM), and affinity maturation of antibodies. Nine-week-old female Hspa13 fl/fl (control) and CD19 cre Hspa13 fl/fl (Hspa13 cKO) mice (three mice per group) were injected i.p. with 1 × 10 9 SRCs (A–C) or NP-KLH (D,E) on days 0 and 7. On day 21 following SRC stimulation, splenocytes were stained with PerCP-conjugated anti-mouse B220 antibodies and sorted by FACS. Single cells were captured using the 10 X Genomics Full Chromium platform and subjected to RNA- and VDJ-sequencing. (A) Hspa13 cKO reduced SRC-induced PBs. Of the single PBs, 27 (3.49%) and 11 (1.07%) (Ighm + , Ighg1 + , Ighg2b + , Ighg2c + , Ighg3 + , Igha + , or Ighe + Cd3d − Cd3e − Cd3gCd4 − Cd8a − Cd19 + Ptprc + Ms4a1 + Ighd − Bcl6 − Aicda − Prdm1 + Xbp1 + Sdc1 + ) within the splenic B cell population were identified by single-cell RNA-sequencing out of 774 and 1,025 single cells corresponding to CD19 cre Hspa13 fl/fl and Hspa13 fl/fl mice, respectively. (B) Hspa13 cKO reduced SRC-induced antibody CSR. Single cells expressing genes encoding IgD, IgM, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibodies were identified by single-cell VDJ-sequencing. The percentage of different antibody subtypes expressed by single cells out of 734 and 382 antibody-expressing single cells from CD19 cre Hspa13 fl/fl and Hspa13 fl/fl mice, respectively, is shown. (C) Hspa13 cKO reduced SRC-induced antibody SHM. The single antibody gene was determined by single-cell VDJ-sequencing. SHM percentages in the CDR (complementarity-determining region) of the heavy (H) and light (L) chains are based on 382 and 734 antibody genes from Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice, respectively. (D) Hspa13 cKO reduced NP-specific SHM induced by NP-KLH. The distribution of the number of mutations per unique clone (VH186.2 segment) is shown. Numbers refer to 100 individual sequences; three animals per group were analyzed. (E) Hspa13 cKO reduced NP-specific high-affinity clones induced by NP-KLH. On day 21 following NP-KLH stimulation, the percentage of NP high-affinity clones containing the W33L mutation in CDR1 in purified GC B cells of Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice was determined. Each dot corresponds to a single animal (30 unique clones/mouse; Mann-Whitney test; error bars represent s.e.m; *** P
    Figure Legend Snippet: Hspa13 cKO reduced class switch recombination (CSR), somatic hypermutation (SHM), and affinity maturation of antibodies. Nine-week-old female Hspa13 fl/fl (control) and CD19 cre Hspa13 fl/fl (Hspa13 cKO) mice (three mice per group) were injected i.p. with 1 × 10 9 SRCs (A–C) or NP-KLH (D,E) on days 0 and 7. On day 21 following SRC stimulation, splenocytes were stained with PerCP-conjugated anti-mouse B220 antibodies and sorted by FACS. Single cells were captured using the 10 X Genomics Full Chromium platform and subjected to RNA- and VDJ-sequencing. (A) Hspa13 cKO reduced SRC-induced PBs. Of the single PBs, 27 (3.49%) and 11 (1.07%) (Ighm + , Ighg1 + , Ighg2b + , Ighg2c + , Ighg3 + , Igha + , or Ighe + Cd3d − Cd3e − Cd3gCd4 − Cd8a − Cd19 + Ptprc + Ms4a1 + Ighd − Bcl6 − Aicda − Prdm1 + Xbp1 + Sdc1 + ) within the splenic B cell population were identified by single-cell RNA-sequencing out of 774 and 1,025 single cells corresponding to CD19 cre Hspa13 fl/fl and Hspa13 fl/fl mice, respectively. (B) Hspa13 cKO reduced SRC-induced antibody CSR. Single cells expressing genes encoding IgD, IgM, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibodies were identified by single-cell VDJ-sequencing. The percentage of different antibody subtypes expressed by single cells out of 734 and 382 antibody-expressing single cells from CD19 cre Hspa13 fl/fl and Hspa13 fl/fl mice, respectively, is shown. (C) Hspa13 cKO reduced SRC-induced antibody SHM. The single antibody gene was determined by single-cell VDJ-sequencing. SHM percentages in the CDR (complementarity-determining region) of the heavy (H) and light (L) chains are based on 382 and 734 antibody genes from Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice, respectively. (D) Hspa13 cKO reduced NP-specific SHM induced by NP-KLH. The distribution of the number of mutations per unique clone (VH186.2 segment) is shown. Numbers refer to 100 individual sequences; three animals per group were analyzed. (E) Hspa13 cKO reduced NP-specific high-affinity clones induced by NP-KLH. On day 21 following NP-KLH stimulation, the percentage of NP high-affinity clones containing the W33L mutation in CDR1 in purified GC B cells of Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice was determined. Each dot corresponds to a single animal (30 unique clones/mouse; Mann-Whitney test; error bars represent s.e.m; *** P

    Techniques Used: Mouse Assay, Injection, Staining, FACS, Sequencing, RNA Sequencing Assay, Expressing, Clone Assay, Mutagenesis, Purification, MANN-WHITNEY

    Hspa13 cKO reduced autoantibodies and proteinuria in pristane-induced lupus and lupus-prone MRL/lpr mouse model. (A) Hspa13 cKO reduced autoantibodies in pristane-induced lupus mice. To induce lupus, 9-week-old female Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice on a B6 background were injected i.p. with 0.5 ml of pristane. On day 21 following pristane stimulation, sera were collected and dsDNA-specific IgM and IgG antibody levels were analyzed by ELISA. (B) Hspa13 cKO reduced proteinuria in the pristane-induced lupus mice. On day 21 following pristane stimulation, urine was collected and proteinuria was measured. (C) Hspa13 cKO reduced autoantibodies in a lupus-prone MRL/lpr mouse model. Sera were collected from Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice in lupus-prone MRL/lpr mice background at 6 months of age and dsDNA-specific IgM and IgG antibody levels were analyzed by ELISA. (D) Hspa13 cKO reduced proteinuria in the lupus-prone MRL/lpr mouse model. Urine was collected from Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice in a lupus-prone MRL/lpr mice background at 6 months of age and proteinuria was measured. (A–D) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P
    Figure Legend Snippet: Hspa13 cKO reduced autoantibodies and proteinuria in pristane-induced lupus and lupus-prone MRL/lpr mouse model. (A) Hspa13 cKO reduced autoantibodies in pristane-induced lupus mice. To induce lupus, 9-week-old female Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice on a B6 background were injected i.p. with 0.5 ml of pristane. On day 21 following pristane stimulation, sera were collected and dsDNA-specific IgM and IgG antibody levels were analyzed by ELISA. (B) Hspa13 cKO reduced proteinuria in the pristane-induced lupus mice. On day 21 following pristane stimulation, urine was collected and proteinuria was measured. (C) Hspa13 cKO reduced autoantibodies in a lupus-prone MRL/lpr mouse model. Sera were collected from Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice in lupus-prone MRL/lpr mice background at 6 months of age and dsDNA-specific IgM and IgG antibody levels were analyzed by ELISA. (D) Hspa13 cKO reduced proteinuria in the lupus-prone MRL/lpr mouse model. Urine was collected from Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice in a lupus-prone MRL/lpr mice background at 6 months of age and proteinuria was measured. (A–D) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P

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

    PBs, PCs, and antibodies were reduced in CD19 cre Hspa13 fl/fl (Hspa13 cKO) mice. (A) Hspa13 cKO did not affect naïve B cells or germinal center (GC) B cells in mice. Splenic lymphocytes from 9-week-old wild type, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were separated using a lymphocyte separation solution; stained with isotype control antibodies, anti-mouse CD19, B220, CD38, and GL7 antibodies; and then analyzed by FACS. The percentages (left panel) and the absolute numbers (right panel) of CD19 + B220 + B cells and CD38 lo GL7 hi B220 + CD19 + GC B cells are shown. (B) Hspa13 cKO reduced PBs, early PCs, and mature PCs in mice. Lymphocytes from the spleen, LNs, and BMs of 9-week-old WT, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were separated using a lymphocyte separation solution; stained with isotype control antibodies, anti-mouse TACI, CD19, B220, and CD138 antibodies; and then analyzed by FACS. The percentages (left panel) and the absolute numbers (right panel) of TACI + CD138 + B220 int CD19 int PBs, TACI + CD138 + B220 − CD19 int early PCs, and TACI + CD138 + B220 − CD19 − mature PCs are shown. (C) Hspa13 cKO reduced antibodies in mice. Sera were collected from 9-week-old WT, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice, and the total IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibody levels were analyzed by ELISA. (A–C) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). * P
    Figure Legend Snippet: PBs, PCs, and antibodies were reduced in CD19 cre Hspa13 fl/fl (Hspa13 cKO) mice. (A) Hspa13 cKO did not affect naïve B cells or germinal center (GC) B cells in mice. Splenic lymphocytes from 9-week-old wild type, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were separated using a lymphocyte separation solution; stained with isotype control antibodies, anti-mouse CD19, B220, CD38, and GL7 antibodies; and then analyzed by FACS. The percentages (left panel) and the absolute numbers (right panel) of CD19 + B220 + B cells and CD38 lo GL7 hi B220 + CD19 + GC B cells are shown. (B) Hspa13 cKO reduced PBs, early PCs, and mature PCs in mice. Lymphocytes from the spleen, LNs, and BMs of 9-week-old WT, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were separated using a lymphocyte separation solution; stained with isotype control antibodies, anti-mouse TACI, CD19, B220, and CD138 antibodies; and then analyzed by FACS. The percentages (left panel) and the absolute numbers (right panel) of TACI + CD138 + B220 int CD19 int PBs, TACI + CD138 + B220 − CD19 int early PCs, and TACI + CD138 + B220 − CD19 − mature PCs are shown. (C) Hspa13 cKO reduced antibodies in mice. Sera were collected from 9-week-old WT, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice, and the total IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibody levels were analyzed by ELISA. (A–C) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). * P

    Techniques Used: Mouse Assay, Staining, FACS, Enzyme-linked Immunosorbent Assay

    Hspa13 cKO reduced the production of LPS-induced PBs, PCs, and antibodies. Splenic B cells from 9-week-old Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were sorted with B220 microbeads and were then stimulated with 10 μg/ml LPS for 0, 1, 2, and 3 days. (A) Hspa13 cKO did not affect LPS-stimulated B-cell proliferation. On days 0, 1, 2, and 3 following LPS stimulation, a CCK8 assay was used to evaluate the cell proliferation. (B,C) Hspa13 cKO did not affect LPS-stimulated B-cell activation. On day 3 following LPS stimulation, cells were stained with isotype control antibodies, anti-mouse B220 and GL7 antibodies, and analyzed by FACS. The percentages (B) and the absolute numbers (C) of B220 + GL7 + B cells are shown. (D,E) Hspa13 cKO reduced LPS-induced PBs, early PCs, and mature PCs. On day 3 following LPS stimulation, cells were stained with isotype control antibodies, anti-mouse TACI, CD19, B220, and CD138 antibodies, and were then analyzed by FACS. The percentages (D) and the absolute numbers (E) of TACI + CD138 + B220 int CD19 int PBs, TACI + CD138 + B220 − CD19 int early PCs, and TACI + CD138 + B220 − CD19 − mature PCs are shown. (F) Hspa13 cKO reduced LPS-induced antibody secretion. On day 3 following LPS stimulation, culture supernatants were collected and the total IgM, IgG1, IgG2b, IgG2c, and IgG3 antibody levels were analyzed by ELISA. (A–F) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by two-way (A) and one-way (C,E,F) ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). *** P
    Figure Legend Snippet: Hspa13 cKO reduced the production of LPS-induced PBs, PCs, and antibodies. Splenic B cells from 9-week-old Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were sorted with B220 microbeads and were then stimulated with 10 μg/ml LPS for 0, 1, 2, and 3 days. (A) Hspa13 cKO did not affect LPS-stimulated B-cell proliferation. On days 0, 1, 2, and 3 following LPS stimulation, a CCK8 assay was used to evaluate the cell proliferation. (B,C) Hspa13 cKO did not affect LPS-stimulated B-cell activation. On day 3 following LPS stimulation, cells were stained with isotype control antibodies, anti-mouse B220 and GL7 antibodies, and analyzed by FACS. The percentages (B) and the absolute numbers (C) of B220 + GL7 + B cells are shown. (D,E) Hspa13 cKO reduced LPS-induced PBs, early PCs, and mature PCs. On day 3 following LPS stimulation, cells were stained with isotype control antibodies, anti-mouse TACI, CD19, B220, and CD138 antibodies, and were then analyzed by FACS. The percentages (D) and the absolute numbers (E) of TACI + CD138 + B220 int CD19 int PBs, TACI + CD138 + B220 − CD19 int early PCs, and TACI + CD138 + B220 − CD19 − mature PCs are shown. (F) Hspa13 cKO reduced LPS-induced antibody secretion. On day 3 following LPS stimulation, culture supernatants were collected and the total IgM, IgG1, IgG2b, IgG2c, and IgG3 antibody levels were analyzed by ELISA. (A–F) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by two-way (A) and one-way (C,E,F) ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). *** P

    Techniques Used: Mouse Assay, CCK-8 Assay, Activation Assay, Staining, FACS, Enzyme-linked Immunosorbent Assay

    Hspa13 cKO reduced 4-hydroxy-3-nitrophenylacetyl (NP)-specific antibody production. Nine-week-old Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were injected i.p. with T cell-independent antigen NP-Ficoll (A) and T cell-dependent antigen NP-keyhole lymphocyte hemocyanin (KLH) (B) on days 0 and 7. On day 21 following NP-Ficoll (A) or NP-KLH (B) stimulation, sera were collected and the NP-specific IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibody levels were analyzed by ELISA. (A,B) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). * P
    Figure Legend Snippet: Hspa13 cKO reduced 4-hydroxy-3-nitrophenylacetyl (NP)-specific antibody production. Nine-week-old Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were injected i.p. with T cell-independent antigen NP-Ficoll (A) and T cell-dependent antigen NP-keyhole lymphocyte hemocyanin (KLH) (B) on days 0 and 7. On day 21 following NP-Ficoll (A) or NP-KLH (B) stimulation, sera were collected and the NP-specific IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibody levels were analyzed by ELISA. (A,B) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). * P

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

    23) Product Images from "Critical role of activation induced cytidine deaminase in Experimental Autoimmune Encephalomyelitis"

    Article Title: Critical role of activation induced cytidine deaminase in Experimental Autoimmune Encephalomyelitis

    Journal: Autoimmunity

    doi: 10.3109/08916934.2012.750301

    Indirect fluorescence for binding of serum antibody to normal brain tissue. Serum from EAE affected WT mice show diffuse, strong white matter associated signal consistent with the presence of MOG specific IgG1. By contrast, corresponding signal is absent using serum from MOG induced KO mice, or healthy controls. There is no difference of signal intensity or distribution for IgM binding to brain, regardless of MOG induction or genotype. Representative images are shown.
    Figure Legend Snippet: Indirect fluorescence for binding of serum antibody to normal brain tissue. Serum from EAE affected WT mice show diffuse, strong white matter associated signal consistent with the presence of MOG specific IgG1. By contrast, corresponding signal is absent using serum from MOG induced KO mice, or healthy controls. There is no difference of signal intensity or distribution for IgM binding to brain, regardless of MOG induction or genotype. Representative images are shown.

    Techniques Used: Fluorescence, Binding Assay, Mouse Assay

    Generation and functional characterization of Aicda deficient mice. A) Gene targeting strategy used to delete the catalytic domain (exon-3) of Aicda locus. The structure of the AID domains is shown in top. The location of the homology arms in the targeting vector is indicated. The schematic of targeting construct used to delete the catalytic domain of AID (exon-3) is shown in the middle. The solid triangles represent Cre recombination sites and the Neomycin cassette was subsequently removed by expression of Cre-recombinase. Representative WT or KO PCR products using 3′ or 5′ arm analysis at the Aicda locus is shown in the right panel. The size of Aicda KO PCR product is indicated and corresponds to the modified locus. Notice that one primer is located in the Neomycin cassette and therefore only detects the targeted Aicda locus. B) FACS analysis of LPS/IL4 stimulated WT or AID KO B cells in vitro. Percentage of IgM or IgG1 are indicated in each quadrant. B220-FITC is used as a B-cell marker.
    Figure Legend Snippet: Generation and functional characterization of Aicda deficient mice. A) Gene targeting strategy used to delete the catalytic domain (exon-3) of Aicda locus. The structure of the AID domains is shown in top. The location of the homology arms in the targeting vector is indicated. The schematic of targeting construct used to delete the catalytic domain of AID (exon-3) is shown in the middle. The solid triangles represent Cre recombination sites and the Neomycin cassette was subsequently removed by expression of Cre-recombinase. Representative WT or KO PCR products using 3′ or 5′ arm analysis at the Aicda locus is shown in the right panel. The size of Aicda KO PCR product is indicated and corresponds to the modified locus. Notice that one primer is located in the Neomycin cassette and therefore only detects the targeted Aicda locus. B) FACS analysis of LPS/IL4 stimulated WT or AID KO B cells in vitro. Percentage of IgM or IgG1 are indicated in each quadrant. B220-FITC is used as a B-cell marker.

    Techniques Used: Functional Assay, Mouse Assay, Plasmid Preparation, Construct, Expressing, Polymerase Chain Reaction, Modification, FACS, In Vitro, Marker

    Characterization of anti-MOG antibodies using ELISA at day zero and 28. A) IgG anti-rhMOG, B) IgM anti-rhMOG, C) IgG anti-MOG peptide 35–55, D) IgM anti-MOG peptide 35–55. Mixed day 28 sera of WT or KO immunized mice were used as standard (arbitrarily defined as 100 U) for IgG or IgM antibodies, respectively. E) IgM binding to rhMOG coated at different concentrations. Sera from 8 mice were pooled for each of the following sample sets: Day 0 WT, Day 0 KO, Day 28 WT, and Day 28 KO. Pooled mice sera IgM Abs normalized at 0.1 mg/ml in the assay. Anti-mouse IgM was used as detection to exclusively measure the binding of IgM antibodies in serum.
    Figure Legend Snippet: Characterization of anti-MOG antibodies using ELISA at day zero and 28. A) IgG anti-rhMOG, B) IgM anti-rhMOG, C) IgG anti-MOG peptide 35–55, D) IgM anti-MOG peptide 35–55. Mixed day 28 sera of WT or KO immunized mice were used as standard (arbitrarily defined as 100 U) for IgG or IgM antibodies, respectively. E) IgM binding to rhMOG coated at different concentrations. Sera from 8 mice were pooled for each of the following sample sets: Day 0 WT, Day 0 KO, Day 28 WT, and Day 28 KO. Pooled mice sera IgM Abs normalized at 0.1 mg/ml in the assay. Anti-mouse IgM was used as detection to exclusively measure the binding of IgM antibodies in serum.

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

    Related Articles

    Immunohistochemistry:

    Article Title: Independent modes of disease repair by AIM protein distinguished in AIM-felinized mice
    Article Snippet: .. Primary antibodies: AIM (rab2 rabbit polyclonal for mice AIM in western blotting and immunohistochemistry, PAC-11 rabbit polyclonal for feline AIM in western blotting, and clone #33 mouse monoclonal for feline AIM in western blotting and immunohistochemistry; established in our laboratory), IgM (A21044, goat polyclonal, Alexa 594 conjugated, Thermo Fisher Scientific, Waltham, MA), F4/80 (clone: BM8 for immunohistochemistry; Thermo Fisher Scientific), CD36 (clone: MF3 for immunohistochemistry; Abcam, Cambridge, UK) and gp73 (goat polyclonal for immunohistochemistry; Santa Cruz, Dallas, TX). .. Secondary Antibodies: goat anti-rabbit IgG (H + L) horseradish peroxidase (HRP) conjugated (Thermo Fisher Scientific), Alexa Fluor 488- or 594-conjugated anti-rat IgG, and Alexa Fluor 647-conjugated anti-rabbit IgG (Thermo Fisher Scientific).

    Mouse Assay:

    Article Title: Independent modes of disease repair by AIM protein distinguished in AIM-felinized mice
    Article Snippet: .. Primary antibodies: AIM (rab2 rabbit polyclonal for mice AIM in western blotting and immunohistochemistry, PAC-11 rabbit polyclonal for feline AIM in western blotting, and clone #33 mouse monoclonal for feline AIM in western blotting and immunohistochemistry; established in our laboratory), IgM (A21044, goat polyclonal, Alexa 594 conjugated, Thermo Fisher Scientific, Waltham, MA), F4/80 (clone: BM8 for immunohistochemistry; Thermo Fisher Scientific), CD36 (clone: MF3 for immunohistochemistry; Abcam, Cambridge, UK) and gp73 (goat polyclonal for immunohistochemistry; Santa Cruz, Dallas, TX). .. Secondary Antibodies: goat anti-rabbit IgG (H + L) horseradish peroxidase (HRP) conjugated (Thermo Fisher Scientific), Alexa Fluor 488- or 594-conjugated anti-rat IgG, and Alexa Fluor 647-conjugated anti-rabbit IgG (Thermo Fisher Scientific).

    Incubation:

    Article Title: Modulation of Receptor Protein Tyrosine Phosphatase Sigma Increases Chondroitin Sulfate Proteoglycan Degradation through Cathepsin B Secretion to Enhance Axon Outgrowth
    Article Snippet: .. Corresponding secondary antibodies conjugated with fluorophores were then used at overnight, 4°C incubation following 3 × 15 min 1 × PBS washes: anti-mouse IgM AlexaFluor-546 (1:500, A21045, Invitrogen), anti-mouse IgG2b AlexaFluor-488 (1:500, , Invitrogen). .. Coverslips were then mounted on slides with Citifluor (17971-25, EM Sciences).

    Article Title: Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody
    Article Snippet: .. Then, cells were incubated with a primary mouse anti-HS antibody (US Biological, cat# H1890, clone 10E4) or a primary mouse anti-CS antibody (Abcam, cat# ab11570, clone CS56), followed by a secondary Alexa Fluor 488-conjugated goat anti-mouse IgM/μ chain-specific antibody (Invitrogen, cat# A21042). .. Finally, cells were examined by flow cytometry analysis.

    Article Title: Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody
    Article Snippet: .. After washing, cells were incubated with a secondary antibody mixture comprising TRITC-conjugated goat anti-rabbit IgG (Sigma, cat# T6778) and an Alexa Fluor 488-conjugated goat anti-mouse IgM/μ chain-specific antibody (Invitrogen, cat# A21042) diluted in surface buffer for 1–2 h at RT. .. Each incubation step was followed by three washes with cold PBS.

    other:

    Article Title: 4-AMINOPYRIDINE ATTENUATES MUSCLE ATROPHY AFTER SCIATIC NERVE CRUSH INJURY IN MICE
    Article Snippet: Additional Materials UsedAntibodies used were anti-Pax7 (mouse IgG1, dilution 1:100, catalog No. AB_528428; Developmental Studies Hybridoma Bank, Iowa City, Iowa), antibody of laminin (rat or rabbit, dilution 1:1,500, catalog No. L0663 or L939; Sigma-Aldrich), anti-Ki67 (dilution 1:400, catalog No. 9129S; Cell Signaling Technology, Danvers, Massachusetts), anti-myogenin (F5D, dilution 1:100, sc-12732; Santa Cruz Bio-technology, Dallas, Texas), anti-GAPDH (dilution 1:2,500, ab9485; Abcam, Cambridge, Massachusetts), AffiniPure Fab fragment goat anti-mouse IgG (H + L, AFFGAI, 0.1 mg/ml; catalog No. 115–007-003; Jackson ImmunoResearch), AlexaFluor 594-conjugated goat anti-mouse IgG (H + L, dilution 1:1,500, catalog No. A-11032,; Life Technologies, Grand Island, New York), AlexaFluor 488-conjugated goat anti-mouse IgM (dilution 1:1,500, catalog No. A-21042; Life Technologies), and AlexaFluor 647-conjugated goat anti-rabbit or anti-mouse antibodies (dilution 1:1,500, catalog No. A-21244 or A-21235; Life Technologies).

    Western Blot:

    Article Title: Independent modes of disease repair by AIM protein distinguished in AIM-felinized mice
    Article Snippet: .. Primary antibodies: AIM (rab2 rabbit polyclonal for mice AIM in western blotting and immunohistochemistry, PAC-11 rabbit polyclonal for feline AIM in western blotting, and clone #33 mouse monoclonal for feline AIM in western blotting and immunohistochemistry; established in our laboratory), IgM (A21044, goat polyclonal, Alexa 594 conjugated, Thermo Fisher Scientific, Waltham, MA), F4/80 (clone: BM8 for immunohistochemistry; Thermo Fisher Scientific), CD36 (clone: MF3 for immunohistochemistry; Abcam, Cambridge, UK) and gp73 (goat polyclonal for immunohistochemistry; Santa Cruz, Dallas, TX). .. Secondary Antibodies: goat anti-rabbit IgG (H + L) horseradish peroxidase (HRP) conjugated (Thermo Fisher Scientific), Alexa Fluor 488- or 594-conjugated anti-rat IgG, and Alexa Fluor 647-conjugated anti-rabbit IgG (Thermo Fisher Scientific).

    Staining:

    Article Title: The Ox40/Ox40 Ligand Pathway Promotes Pathogenic Th Cell Responses, Plasmablast Accumulation, and Lupus Nephritis in NZB/W F1 Mice
    Article Snippet: .. Glomerular IC deposits were visualized on 5-μm acetone-fixed OCT-embedded kidney sections by direct immunofluorescence staining using Alexa Fluor 488–conjugated donkey anti-mouse IgG (cat. no. A-21202; Invitrogen), goat anti-mouse IgM (cat. no. A-21042; Invitrogen), or fluorescein-conjugated goat anti-complement C3 (cat. no. 55510; MP Biomedicals). .. Naive donkey or goat IgG (Jackson ImmunoResearch) was used in parallel as nonspecific isotype controls and were negative.

    Immunofluorescence:

    Article Title: The Ox40/Ox40 Ligand Pathway Promotes Pathogenic Th Cell Responses, Plasmablast Accumulation, and Lupus Nephritis in NZB/W F1 Mice
    Article Snippet: .. Glomerular IC deposits were visualized on 5-μm acetone-fixed OCT-embedded kidney sections by direct immunofluorescence staining using Alexa Fluor 488–conjugated donkey anti-mouse IgG (cat. no. A-21202; Invitrogen), goat anti-mouse IgM (cat. no. A-21042; Invitrogen), or fluorescein-conjugated goat anti-complement C3 (cat. no. 55510; MP Biomedicals). .. Naive donkey or goat IgG (Jackson ImmunoResearch) was used in parallel as nonspecific isotype controls and were negative.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher goat anti mouse igm heavy chain cross adsorbed secondary antibody
    States of inflammation and fibrosis in the liver in the absence of serum <t>IgM-free</t> <t>AIM.</t> ( a ) The mRNA levels of various genes responsive to different types of stresses (i.e., endoplasmic reticulum, mitochondrial, or oxidative stress) addressed by qPCR using RNA from the whole liver of AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 5–6 for 12-week HFD per group). Error bar indicates the SEM. (b,c) The mRNA levels of inflammatory cytokine genes in the liver, as assessed by qPCR using the RNA as in ( a ). (d ) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsμ mice fed an HFD for 12 weeks stained with Sirius red Stain. Scale bars, 100 μm. (e) The mRNA levels of fibrogenetic genes in the liver, as assessed by qPCR using the same RNA as in ( a ). In (a – c) and (e) , *is used to represent the statistical significance between the values of each mouse strain group within the same period, whereas # is attached to the bar of 12 w when the value of 12 w was significantly changed compared with that of 0 w in the same mouse strain group.
    Goat Anti Mouse Igm Heavy Chain Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti mouse igm heavy chain cross adsorbed secondary antibody/product/Thermo Fisher
    Average 99 stars, based on 30 article reviews
    Price from $9.99 to $1999.99
    goat anti mouse igm heavy chain cross adsorbed secondary antibody - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher igm
    Increased NP-specific Ab in immunized Eaf2 −/− mice. ( a ) Five pairs of WT and Eaf2 −/− mice were immunized with 10 μg of NP–CGG in alum. Serum levels of NP-specific <t>IgG</t> 1 Ab were measured with NP25-BSA (upper) and NP2-BSA (lower) at the indicated time points as described in Methods section. ( b ) AFC in spleen (left) and BM (right) at 6 weeks after NP–CGG immunization. NP2 and NP25 detect high affinity and total NP-specific AFC, respectively. ( c ) NP-specific <t>IgM</t> (upper) and IgG 3 (lower) serum Ab levels after immunization with NP-Ficoll. The results of five pairs of WT and Eaf2 −/− mice are shown. ( d ) NP-specific IgM AFC in the spleen and BM at 21 weeks after NP-Ficoll immunization. Three pairs of WT and Eaf2 −/− mice were analysed. ( e ) and ( f ), Rag1 −/− mice reconstituted with WT T+WT B or WT T+ Eaf2 −/− B cells were immunized with NP–CGG and analysed for ( e ) NP-specific total IgG 1 Ab (NP25) and high-affinity IgG 1 Ab (NP2) in the serum and ( f ) AFC in the spleen. Open circles, Rag1 −/− mice reconstituted with WT T+WT B cells; Solid circles, Rag1 −/− mice reconstituted with WT T+ Eaf2 −/− B cells. Arrows in a and c indicate immunization times. The red bar indicates the mean value of each group. * P
    Igm, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 397 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/igm/product/Thermo Fisher
    Average 99 stars, based on 397 article reviews
    Price from $9.99 to $1999.99
    igm - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    Image Search Results


    States of inflammation and fibrosis in the liver in the absence of serum IgM-free AIM. ( a ) The mRNA levels of various genes responsive to different types of stresses (i.e., endoplasmic reticulum, mitochondrial, or oxidative stress) addressed by qPCR using RNA from the whole liver of AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 5–6 for 12-week HFD per group). Error bar indicates the SEM. (b,c) The mRNA levels of inflammatory cytokine genes in the liver, as assessed by qPCR using the RNA as in ( a ). (d ) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsμ mice fed an HFD for 12 weeks stained with Sirius red Stain. Scale bars, 100 μm. (e) The mRNA levels of fibrogenetic genes in the liver, as assessed by qPCR using the same RNA as in ( a ). In (a – c) and (e) , *is used to represent the statistical significance between the values of each mouse strain group within the same period, whereas # is attached to the bar of 12 w when the value of 12 w was significantly changed compared with that of 0 w in the same mouse strain group.

    Journal: Scientific Reports

    Article Title: Independent modes of disease repair by AIM protein distinguished in AIM-felinized mice

    doi: 10.1038/s41598-018-31580-6

    Figure Lengend Snippet: States of inflammation and fibrosis in the liver in the absence of serum IgM-free AIM. ( a ) The mRNA levels of various genes responsive to different types of stresses (i.e., endoplasmic reticulum, mitochondrial, or oxidative stress) addressed by qPCR using RNA from the whole liver of AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 5–6 for 12-week HFD per group). Error bar indicates the SEM. (b,c) The mRNA levels of inflammatory cytokine genes in the liver, as assessed by qPCR using the RNA as in ( a ). (d ) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsμ mice fed an HFD for 12 weeks stained with Sirius red Stain. Scale bars, 100 μm. (e) The mRNA levels of fibrogenetic genes in the liver, as assessed by qPCR using the same RNA as in ( a ). In (a – c) and (e) , *is used to represent the statistical significance between the values of each mouse strain group within the same period, whereas # is attached to the bar of 12 w when the value of 12 w was significantly changed compared with that of 0 w in the same mouse strain group.

    Article Snippet: Primary antibodies: AIM (rab2 rabbit polyclonal for mice AIM in western blotting and immunohistochemistry, PAC-11 rabbit polyclonal for feline AIM in western blotting, and clone #33 mouse monoclonal for feline AIM in western blotting and immunohistochemistry; established in our laboratory), IgM (A21044, goat polyclonal, Alexa 594 conjugated, Thermo Fisher Scientific, Waltham, MA), F4/80 (clone: BM8 for immunohistochemistry; Thermo Fisher Scientific), CD36 (clone: MF3 for immunohistochemistry; Abcam, Cambridge, UK) and gp73 (goat polyclonal for immunohistochemistry; Santa Cruz, Dallas, TX).

    Techniques: Real-time Polymerase Chain Reaction, Mouse Assay, Staining

    Effect of serum IgM-free AIM on obesity and liver steatosis. ( a,b) Weights from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM. (a) Body weights. (b ) Weights of epidydimal adipose tissues. (c) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Adipocyte sizes of 50 independent adipocytes in different areas were evaluated. Results are presented as average ± SEM (in μm 2 ). Scale bars, 100 μm. ( d ) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, and AIM −/− mice (fed an HFD for 12 weeks) stained for AIM (blue), F4/80 (macrophage marker; green), and IgM (red, WT and AIM −/− mice. See Supplementary Fig. 1 for AIM-felinized mice). Yellow arrows represent where IgM-free AIM signals exist, while red arrows indicate where AIM is co-stained with IgM. Scale bars, 100 μm. (e) The mRNA levels of AIM and F4/80 were assessed by qPCR using RNA isolated from epididymal fat in WT mice before or after being fed an HFD for 12 weeks. Values were normalized to those of GAPDH and presented as the expression relative to that of AIM from lean WT mice liver and of F4/80 from fat tissues before being fed an HFD (n = 4 per group). Error bar indicates the SEM. (f) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Scale bars, 100 µm. (g) Liver weights and TG contents from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM.

    Journal: Scientific Reports

    Article Title: Independent modes of disease repair by AIM protein distinguished in AIM-felinized mice

    doi: 10.1038/s41598-018-31580-6

    Figure Lengend Snippet: Effect of serum IgM-free AIM on obesity and liver steatosis. ( a,b) Weights from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM. (a) Body weights. (b ) Weights of epidydimal adipose tissues. (c) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Adipocyte sizes of 50 independent adipocytes in different areas were evaluated. Results are presented as average ± SEM (in μm 2 ). Scale bars, 100 μm. ( d ) Representative photomicrographs of epididymal fat tissues from AIM-felinized, WT, and AIM −/− mice (fed an HFD for 12 weeks) stained for AIM (blue), F4/80 (macrophage marker; green), and IgM (red, WT and AIM −/− mice. See Supplementary Fig. 1 for AIM-felinized mice). Yellow arrows represent where IgM-free AIM signals exist, while red arrows indicate where AIM is co-stained with IgM. Scale bars, 100 μm. (e) The mRNA levels of AIM and F4/80 were assessed by qPCR using RNA isolated from epididymal fat in WT mice before or after being fed an HFD for 12 weeks. Values were normalized to those of GAPDH and presented as the expression relative to that of AIM from lean WT mice liver and of F4/80 from fat tissues before being fed an HFD (n = 4 per group). Error bar indicates the SEM. (f) Representative photomicrographs of liver from AIM-felinized, WT, AIM −/− , and Δsµ mice fed an HFD for 12 weeks stained with H E. Scale bars, 100 µm. (g) Liver weights and TG contents from AIM-felinized, WT, AIM −/− , and Δsµ mice before and after being fed an HFD for 12 weeks (n = 4 before HFD and n = 6–9 for 12-week HFD per group). Error bar indicates the SEM.

    Article Snippet: Primary antibodies: AIM (rab2 rabbit polyclonal for mice AIM in western blotting and immunohistochemistry, PAC-11 rabbit polyclonal for feline AIM in western blotting, and clone #33 mouse monoclonal for feline AIM in western blotting and immunohistochemistry; established in our laboratory), IgM (A21044, goat polyclonal, Alexa 594 conjugated, Thermo Fisher Scientific, Waltham, MA), F4/80 (clone: BM8 for immunohistochemistry; Thermo Fisher Scientific), CD36 (clone: MF3 for immunohistochemistry; Abcam, Cambridge, UK) and gp73 (goat polyclonal for immunohistochemistry; Santa Cruz, Dallas, TX).

    Techniques: Mouse Assay, Staining, Marker, Real-time Polymerase Chain Reaction, Isolation, Expressing

    Increased NP-specific Ab in immunized Eaf2 −/− mice. ( a ) Five pairs of WT and Eaf2 −/− mice were immunized with 10 μg of NP–CGG in alum. Serum levels of NP-specific IgG 1 Ab were measured with NP25-BSA (upper) and NP2-BSA (lower) at the indicated time points as described in Methods section. ( b ) AFC in spleen (left) and BM (right) at 6 weeks after NP–CGG immunization. NP2 and NP25 detect high affinity and total NP-specific AFC, respectively. ( c ) NP-specific IgM (upper) and IgG 3 (lower) serum Ab levels after immunization with NP-Ficoll. The results of five pairs of WT and Eaf2 −/− mice are shown. ( d ) NP-specific IgM AFC in the spleen and BM at 21 weeks after NP-Ficoll immunization. Three pairs of WT and Eaf2 −/− mice were analysed. ( e ) and ( f ), Rag1 −/− mice reconstituted with WT T+WT B or WT T+ Eaf2 −/− B cells were immunized with NP–CGG and analysed for ( e ) NP-specific total IgG 1 Ab (NP25) and high-affinity IgG 1 Ab (NP2) in the serum and ( f ) AFC in the spleen. Open circles, Rag1 −/− mice reconstituted with WT T+WT B cells; Solid circles, Rag1 −/− mice reconstituted with WT T+ Eaf2 −/− B cells. Arrows in a and c indicate immunization times. The red bar indicates the mean value of each group. * P

    Journal: Nature Communications

    Article Title: EAF2 mediates germinal centre B-cell apoptosis to suppress excessive immune responses and prevent autoimmunity

    doi: 10.1038/ncomms10836

    Figure Lengend Snippet: Increased NP-specific Ab in immunized Eaf2 −/− mice. ( a ) Five pairs of WT and Eaf2 −/− mice were immunized with 10 μg of NP–CGG in alum. Serum levels of NP-specific IgG 1 Ab were measured with NP25-BSA (upper) and NP2-BSA (lower) at the indicated time points as described in Methods section. ( b ) AFC in spleen (left) and BM (right) at 6 weeks after NP–CGG immunization. NP2 and NP25 detect high affinity and total NP-specific AFC, respectively. ( c ) NP-specific IgM (upper) and IgG 3 (lower) serum Ab levels after immunization with NP-Ficoll. The results of five pairs of WT and Eaf2 −/− mice are shown. ( d ) NP-specific IgM AFC in the spleen and BM at 21 weeks after NP-Ficoll immunization. Three pairs of WT and Eaf2 −/− mice were analysed. ( e ) and ( f ), Rag1 −/− mice reconstituted with WT T+WT B or WT T+ Eaf2 −/− B cells were immunized with NP–CGG and analysed for ( e ) NP-specific total IgG 1 Ab (NP25) and high-affinity IgG 1 Ab (NP2) in the serum and ( f ) AFC in the spleen. Open circles, Rag1 −/− mice reconstituted with WT T+WT B cells; Solid circles, Rag1 −/− mice reconstituted with WT T+ Eaf2 −/− B cells. Arrows in a and c indicate immunization times. The red bar indicates the mean value of each group. * P

    Article Snippet: After washing, HRP-conjugated goat anti-mouse IgG, IgM, IgG1 or IgG2a Ab were added and incubated for 1 h at room temperature, and 1-Step Ultra TMB ELISA substrate (34028, Thermo) was added to each well.

    Techniques: Mouse Assay

    Hspa13 cKO reduced sheep red blood cell (SRC)-induced PB/PC and antibody production. Nine-week-old female Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were injected intraperitoneally (i.p.) with 1 × 10 9 SRCs on days 0 and 7. (A,B) Hspa13 cKO did not affect SRC-induce GC B-cell production. On day 21 following SRC stimulation, splenic lymphocytes were stained with isotype control antibodies, anti-mouse CD19, B220, CD38, and GL7 antibodies, and were then analyzed by FACS. The percentages (A) and the absolute numbers (B) of CD38 lo GL7 hi GC cells gated on CD19 + B220 + are shown. (C,D) Hspa13 cKO did not affect the SRC-induced dark zone (DZ) and light zone (LZ) GC B-cell production. On day 21 following SRC stimulation, splenic lymphocytes were stained with anti-mouse CD19, B220, CD38, GL7, CXCR4, and CD86 antibodies, and were then analyzed by FACS. The percentages (C) and the absolute numbers (D) of CXCR4 hi CD86 lo DZ and CXCR4 lo CD86 hi LZ GC B cells gated on CD19 + B220 + CD38 lo GL7 hi GC cells are shown. (E,F) Hspa13 cKO reduced SRC-induced IgG1-, IgG2b-, IgG2c-, and IgG3-expressing PBs/PCs. On day 21 following SRC stimulation, splenic lymphocytes were collected and intracellular staining was performed with isotype control antibodies, anti-mouse B220, IgG1, IgG2b, IgG2c, and IgG3 antibodies. The percentages (E) and the absolute numbers (F) of IgG1-, IgG2b-, IgG2c-, and IgG3-expressing B220 + PBs and B220 − PCs are shown. (G) Hspa13 cKO reduced SRC-induced antibody secretion. On day 21 following SRC stimulation, sera were collected and the total IgM, IgG, IgG1, IgG2b, IgG2c, and IgG3 antibody levels were analyzed by ELISA. (A–G) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by two-way (D) and one-way (B,F,G) ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P

    Journal: Frontiers in Immunology

    Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

    doi: 10.3389/fimmu.2020.00913

    Figure Lengend Snippet: Hspa13 cKO reduced sheep red blood cell (SRC)-induced PB/PC and antibody production. Nine-week-old female Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were injected intraperitoneally (i.p.) with 1 × 10 9 SRCs on days 0 and 7. (A,B) Hspa13 cKO did not affect SRC-induce GC B-cell production. On day 21 following SRC stimulation, splenic lymphocytes were stained with isotype control antibodies, anti-mouse CD19, B220, CD38, and GL7 antibodies, and were then analyzed by FACS. The percentages (A) and the absolute numbers (B) of CD38 lo GL7 hi GC cells gated on CD19 + B220 + are shown. (C,D) Hspa13 cKO did not affect the SRC-induced dark zone (DZ) and light zone (LZ) GC B-cell production. On day 21 following SRC stimulation, splenic lymphocytes were stained with anti-mouse CD19, B220, CD38, GL7, CXCR4, and CD86 antibodies, and were then analyzed by FACS. The percentages (C) and the absolute numbers (D) of CXCR4 hi CD86 lo DZ and CXCR4 lo CD86 hi LZ GC B cells gated on CD19 + B220 + CD38 lo GL7 hi GC cells are shown. (E,F) Hspa13 cKO reduced SRC-induced IgG1-, IgG2b-, IgG2c-, and IgG3-expressing PBs/PCs. On day 21 following SRC stimulation, splenic lymphocytes were collected and intracellular staining was performed with isotype control antibodies, anti-mouse B220, IgG1, IgG2b, IgG2c, and IgG3 antibodies. The percentages (E) and the absolute numbers (F) of IgG1-, IgG2b-, IgG2c-, and IgG3-expressing B220 + PBs and B220 − PCs are shown. (G) Hspa13 cKO reduced SRC-induced antibody secretion. On day 21 following SRC stimulation, sera were collected and the total IgM, IgG, IgG1, IgG2b, IgG2c, and IgG3 antibody levels were analyzed by ELISA. (A–G) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by two-way (D) and one-way (B,F,G) ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P

    Article Snippet: Then after washing, 4 μg/ml HRP-conjugated anti-mouse IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, or IgE antibodies were added to the plate and were incubated for another hour at 37°C.

    Techniques: Mouse Assay, Injection, Staining, FACS, Expressing, Enzyme-linked Immunosorbent Assay

    Hspa13 cKO reduced class switch recombination (CSR), somatic hypermutation (SHM), and affinity maturation of antibodies. Nine-week-old female Hspa13 fl/fl (control) and CD19 cre Hspa13 fl/fl (Hspa13 cKO) mice (three mice per group) were injected i.p. with 1 × 10 9 SRCs (A–C) or NP-KLH (D,E) on days 0 and 7. On day 21 following SRC stimulation, splenocytes were stained with PerCP-conjugated anti-mouse B220 antibodies and sorted by FACS. Single cells were captured using the 10 X Genomics Full Chromium platform and subjected to RNA- and VDJ-sequencing. (A) Hspa13 cKO reduced SRC-induced PBs. Of the single PBs, 27 (3.49%) and 11 (1.07%) (Ighm + , Ighg1 + , Ighg2b + , Ighg2c + , Ighg3 + , Igha + , or Ighe + Cd3d − Cd3e − Cd3gCd4 − Cd8a − Cd19 + Ptprc + Ms4a1 + Ighd − Bcl6 − Aicda − Prdm1 + Xbp1 + Sdc1 + ) within the splenic B cell population were identified by single-cell RNA-sequencing out of 774 and 1,025 single cells corresponding to CD19 cre Hspa13 fl/fl and Hspa13 fl/fl mice, respectively. (B) Hspa13 cKO reduced SRC-induced antibody CSR. Single cells expressing genes encoding IgD, IgM, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibodies were identified by single-cell VDJ-sequencing. The percentage of different antibody subtypes expressed by single cells out of 734 and 382 antibody-expressing single cells from CD19 cre Hspa13 fl/fl and Hspa13 fl/fl mice, respectively, is shown. (C) Hspa13 cKO reduced SRC-induced antibody SHM. The single antibody gene was determined by single-cell VDJ-sequencing. SHM percentages in the CDR (complementarity-determining region) of the heavy (H) and light (L) chains are based on 382 and 734 antibody genes from Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice, respectively. (D) Hspa13 cKO reduced NP-specific SHM induced by NP-KLH. The distribution of the number of mutations per unique clone (VH186.2 segment) is shown. Numbers refer to 100 individual sequences; three animals per group were analyzed. (E) Hspa13 cKO reduced NP-specific high-affinity clones induced by NP-KLH. On day 21 following NP-KLH stimulation, the percentage of NP high-affinity clones containing the W33L mutation in CDR1 in purified GC B cells of Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice was determined. Each dot corresponds to a single animal (30 unique clones/mouse; Mann-Whitney test; error bars represent s.e.m; *** P

    Journal: Frontiers in Immunology

    Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

    doi: 10.3389/fimmu.2020.00913

    Figure Lengend Snippet: Hspa13 cKO reduced class switch recombination (CSR), somatic hypermutation (SHM), and affinity maturation of antibodies. Nine-week-old female Hspa13 fl/fl (control) and CD19 cre Hspa13 fl/fl (Hspa13 cKO) mice (three mice per group) were injected i.p. with 1 × 10 9 SRCs (A–C) or NP-KLH (D,E) on days 0 and 7. On day 21 following SRC stimulation, splenocytes were stained with PerCP-conjugated anti-mouse B220 antibodies and sorted by FACS. Single cells were captured using the 10 X Genomics Full Chromium platform and subjected to RNA- and VDJ-sequencing. (A) Hspa13 cKO reduced SRC-induced PBs. Of the single PBs, 27 (3.49%) and 11 (1.07%) (Ighm + , Ighg1 + , Ighg2b + , Ighg2c + , Ighg3 + , Igha + , or Ighe + Cd3d − Cd3e − Cd3gCd4 − Cd8a − Cd19 + Ptprc + Ms4a1 + Ighd − Bcl6 − Aicda − Prdm1 + Xbp1 + Sdc1 + ) within the splenic B cell population were identified by single-cell RNA-sequencing out of 774 and 1,025 single cells corresponding to CD19 cre Hspa13 fl/fl and Hspa13 fl/fl mice, respectively. (B) Hspa13 cKO reduced SRC-induced antibody CSR. Single cells expressing genes encoding IgD, IgM, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibodies were identified by single-cell VDJ-sequencing. The percentage of different antibody subtypes expressed by single cells out of 734 and 382 antibody-expressing single cells from CD19 cre Hspa13 fl/fl and Hspa13 fl/fl mice, respectively, is shown. (C) Hspa13 cKO reduced SRC-induced antibody SHM. The single antibody gene was determined by single-cell VDJ-sequencing. SHM percentages in the CDR (complementarity-determining region) of the heavy (H) and light (L) chains are based on 382 and 734 antibody genes from Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice, respectively. (D) Hspa13 cKO reduced NP-specific SHM induced by NP-KLH. The distribution of the number of mutations per unique clone (VH186.2 segment) is shown. Numbers refer to 100 individual sequences; three animals per group were analyzed. (E) Hspa13 cKO reduced NP-specific high-affinity clones induced by NP-KLH. On day 21 following NP-KLH stimulation, the percentage of NP high-affinity clones containing the W33L mutation in CDR1 in purified GC B cells of Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice was determined. Each dot corresponds to a single animal (30 unique clones/mouse; Mann-Whitney test; error bars represent s.e.m; *** P

    Article Snippet: Then after washing, 4 μg/ml HRP-conjugated anti-mouse IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, or IgE antibodies were added to the plate and were incubated for another hour at 37°C.

    Techniques: Mouse Assay, Injection, Staining, FACS, Sequencing, RNA Sequencing Assay, Expressing, Clone Assay, Mutagenesis, Purification, MANN-WHITNEY

    Hspa13 cKO reduced autoantibodies and proteinuria in pristane-induced lupus and lupus-prone MRL/lpr mouse model. (A) Hspa13 cKO reduced autoantibodies in pristane-induced lupus mice. To induce lupus, 9-week-old female Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice on a B6 background were injected i.p. with 0.5 ml of pristane. On day 21 following pristane stimulation, sera were collected and dsDNA-specific IgM and IgG antibody levels were analyzed by ELISA. (B) Hspa13 cKO reduced proteinuria in the pristane-induced lupus mice. On day 21 following pristane stimulation, urine was collected and proteinuria was measured. (C) Hspa13 cKO reduced autoantibodies in a lupus-prone MRL/lpr mouse model. Sera were collected from Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice in lupus-prone MRL/lpr mice background at 6 months of age and dsDNA-specific IgM and IgG antibody levels were analyzed by ELISA. (D) Hspa13 cKO reduced proteinuria in the lupus-prone MRL/lpr mouse model. Urine was collected from Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice in a lupus-prone MRL/lpr mice background at 6 months of age and proteinuria was measured. (A–D) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P

    Journal: Frontiers in Immunology

    Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

    doi: 10.3389/fimmu.2020.00913

    Figure Lengend Snippet: Hspa13 cKO reduced autoantibodies and proteinuria in pristane-induced lupus and lupus-prone MRL/lpr mouse model. (A) Hspa13 cKO reduced autoantibodies in pristane-induced lupus mice. To induce lupus, 9-week-old female Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice on a B6 background were injected i.p. with 0.5 ml of pristane. On day 21 following pristane stimulation, sera were collected and dsDNA-specific IgM and IgG antibody levels were analyzed by ELISA. (B) Hspa13 cKO reduced proteinuria in the pristane-induced lupus mice. On day 21 following pristane stimulation, urine was collected and proteinuria was measured. (C) Hspa13 cKO reduced autoantibodies in a lupus-prone MRL/lpr mouse model. Sera were collected from Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice in lupus-prone MRL/lpr mice background at 6 months of age and dsDNA-specific IgM and IgG antibody levels were analyzed by ELISA. (D) Hspa13 cKO reduced proteinuria in the lupus-prone MRL/lpr mouse model. Urine was collected from Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice in a lupus-prone MRL/lpr mice background at 6 months of age and proteinuria was measured. (A–D) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P

    Article Snippet: Then after washing, 4 μg/ml HRP-conjugated anti-mouse IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, or IgE antibodies were added to the plate and were incubated for another hour at 37°C.

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

    PBs, PCs, and antibodies were reduced in CD19 cre Hspa13 fl/fl (Hspa13 cKO) mice. (A) Hspa13 cKO did not affect naïve B cells or germinal center (GC) B cells in mice. Splenic lymphocytes from 9-week-old wild type, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were separated using a lymphocyte separation solution; stained with isotype control antibodies, anti-mouse CD19, B220, CD38, and GL7 antibodies; and then analyzed by FACS. The percentages (left panel) and the absolute numbers (right panel) of CD19 + B220 + B cells and CD38 lo GL7 hi B220 + CD19 + GC B cells are shown. (B) Hspa13 cKO reduced PBs, early PCs, and mature PCs in mice. Lymphocytes from the spleen, LNs, and BMs of 9-week-old WT, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were separated using a lymphocyte separation solution; stained with isotype control antibodies, anti-mouse TACI, CD19, B220, and CD138 antibodies; and then analyzed by FACS. The percentages (left panel) and the absolute numbers (right panel) of TACI + CD138 + B220 int CD19 int PBs, TACI + CD138 + B220 − CD19 int early PCs, and TACI + CD138 + B220 − CD19 − mature PCs are shown. (C) Hspa13 cKO reduced antibodies in mice. Sera were collected from 9-week-old WT, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice, and the total IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibody levels were analyzed by ELISA. (A–C) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). * P

    Journal: Frontiers in Immunology

    Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

    doi: 10.3389/fimmu.2020.00913

    Figure Lengend Snippet: PBs, PCs, and antibodies were reduced in CD19 cre Hspa13 fl/fl (Hspa13 cKO) mice. (A) Hspa13 cKO did not affect naïve B cells or germinal center (GC) B cells in mice. Splenic lymphocytes from 9-week-old wild type, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were separated using a lymphocyte separation solution; stained with isotype control antibodies, anti-mouse CD19, B220, CD38, and GL7 antibodies; and then analyzed by FACS. The percentages (left panel) and the absolute numbers (right panel) of CD19 + B220 + B cells and CD38 lo GL7 hi B220 + CD19 + GC B cells are shown. (B) Hspa13 cKO reduced PBs, early PCs, and mature PCs in mice. Lymphocytes from the spleen, LNs, and BMs of 9-week-old WT, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were separated using a lymphocyte separation solution; stained with isotype control antibodies, anti-mouse TACI, CD19, B220, and CD138 antibodies; and then analyzed by FACS. The percentages (left panel) and the absolute numbers (right panel) of TACI + CD138 + B220 int CD19 int PBs, TACI + CD138 + B220 − CD19 int early PCs, and TACI + CD138 + B220 − CD19 − mature PCs are shown. (C) Hspa13 cKO reduced antibodies in mice. Sera were collected from 9-week-old WT, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice, and the total IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibody levels were analyzed by ELISA. (A–C) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). * P

    Article Snippet: Then after washing, 4 μg/ml HRP-conjugated anti-mouse IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, or IgE antibodies were added to the plate and were incubated for another hour at 37°C.

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

    Hspa13 cKO reduced the production of LPS-induced PBs, PCs, and antibodies. Splenic B cells from 9-week-old Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were sorted with B220 microbeads and were then stimulated with 10 μg/ml LPS for 0, 1, 2, and 3 days. (A) Hspa13 cKO did not affect LPS-stimulated B-cell proliferation. On days 0, 1, 2, and 3 following LPS stimulation, a CCK8 assay was used to evaluate the cell proliferation. (B,C) Hspa13 cKO did not affect LPS-stimulated B-cell activation. On day 3 following LPS stimulation, cells were stained with isotype control antibodies, anti-mouse B220 and GL7 antibodies, and analyzed by FACS. The percentages (B) and the absolute numbers (C) of B220 + GL7 + B cells are shown. (D,E) Hspa13 cKO reduced LPS-induced PBs, early PCs, and mature PCs. On day 3 following LPS stimulation, cells were stained with isotype control antibodies, anti-mouse TACI, CD19, B220, and CD138 antibodies, and were then analyzed by FACS. The percentages (D) and the absolute numbers (E) of TACI + CD138 + B220 int CD19 int PBs, TACI + CD138 + B220 − CD19 int early PCs, and TACI + CD138 + B220 − CD19 − mature PCs are shown. (F) Hspa13 cKO reduced LPS-induced antibody secretion. On day 3 following LPS stimulation, culture supernatants were collected and the total IgM, IgG1, IgG2b, IgG2c, and IgG3 antibody levels were analyzed by ELISA. (A–F) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by two-way (A) and one-way (C,E,F) ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). *** P

    Journal: Frontiers in Immunology

    Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

    doi: 10.3389/fimmu.2020.00913

    Figure Lengend Snippet: Hspa13 cKO reduced the production of LPS-induced PBs, PCs, and antibodies. Splenic B cells from 9-week-old Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were sorted with B220 microbeads and were then stimulated with 10 μg/ml LPS for 0, 1, 2, and 3 days. (A) Hspa13 cKO did not affect LPS-stimulated B-cell proliferation. On days 0, 1, 2, and 3 following LPS stimulation, a CCK8 assay was used to evaluate the cell proliferation. (B,C) Hspa13 cKO did not affect LPS-stimulated B-cell activation. On day 3 following LPS stimulation, cells were stained with isotype control antibodies, anti-mouse B220 and GL7 antibodies, and analyzed by FACS. The percentages (B) and the absolute numbers (C) of B220 + GL7 + B cells are shown. (D,E) Hspa13 cKO reduced LPS-induced PBs, early PCs, and mature PCs. On day 3 following LPS stimulation, cells were stained with isotype control antibodies, anti-mouse TACI, CD19, B220, and CD138 antibodies, and were then analyzed by FACS. The percentages (D) and the absolute numbers (E) of TACI + CD138 + B220 int CD19 int PBs, TACI + CD138 + B220 − CD19 int early PCs, and TACI + CD138 + B220 − CD19 − mature PCs are shown. (F) Hspa13 cKO reduced LPS-induced antibody secretion. On day 3 following LPS stimulation, culture supernatants were collected and the total IgM, IgG1, IgG2b, IgG2c, and IgG3 antibody levels were analyzed by ELISA. (A–F) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by two-way (A) and one-way (C,E,F) ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). *** P

    Article Snippet: Then after washing, 4 μg/ml HRP-conjugated anti-mouse IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, or IgE antibodies were added to the plate and were incubated for another hour at 37°C.

    Techniques: Mouse Assay, CCK-8 Assay, Activation Assay, Staining, FACS, Enzyme-linked Immunosorbent Assay

    Hspa13 cKO reduced 4-hydroxy-3-nitrophenylacetyl (NP)-specific antibody production. Nine-week-old Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were injected i.p. with T cell-independent antigen NP-Ficoll (A) and T cell-dependent antigen NP-keyhole lymphocyte hemocyanin (KLH) (B) on days 0 and 7. On day 21 following NP-Ficoll (A) or NP-KLH (B) stimulation, sera were collected and the NP-specific IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibody levels were analyzed by ELISA. (A,B) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). * P

    Journal: Frontiers in Immunology

    Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

    doi: 10.3389/fimmu.2020.00913

    Figure Lengend Snippet: Hspa13 cKO reduced 4-hydroxy-3-nitrophenylacetyl (NP)-specific antibody production. Nine-week-old Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were injected i.p. with T cell-independent antigen NP-Ficoll (A) and T cell-dependent antigen NP-keyhole lymphocyte hemocyanin (KLH) (B) on days 0 and 7. On day 21 following NP-Ficoll (A) or NP-KLH (B) stimulation, sera were collected and the NP-specific IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibody levels were analyzed by ELISA. (A,B) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). * P

    Article Snippet: Then after washing, 4 μg/ml HRP-conjugated anti-mouse IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, or IgE antibodies were added to the plate and were incubated for another hour at 37°C.

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