cd38  (BioLegend)

 
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  • 97
    Name:
    PE Cy7 anti mouse CD38
    Description:
    PE Cy7 anti mouse CD38 90 Isotype Rat IgG2a κ Reactivity Mouse Apps FC Size 25 μg
    Catalog Number:
    102717
    Price:
    90
    Applications:
    FC
    Conjugate:
    PE Cy7
    Immunogen:
    Mouse bone marrow pre-B cells
    Size:
    25 μg
    Category:
    Mouse Immunology
    Preparation:
    The antibody was purified by affinity chromatography and conjugated with PE Cy7 under optimal conditions The solution is free of unconjugated PE Cy7 and unconjugated antibody
    Source:
    Rat
    Quantity:
    1
    Buy from Supplier


    Structured Review

    BioLegend cd38
    PE Cy7 anti mouse CD38
    PE Cy7 anti mouse CD38 90 Isotype Rat IgG2a κ Reactivity Mouse Apps FC Size 25 μg
    https://www.bioz.com/result/cd38/product/BioLegend
    Average 97 stars, based on 96 article reviews
    Price from $9.99 to $1999.99
    cd38 - by Bioz Stars, 2020-09
    97/100 stars

    Images

    1) Product Images from "A potent tetravalent T-cell–engaging bispecific antibody against CD33 in acute myeloid leukemia"

    Article Title: A potent tetravalent T-cell–engaging bispecific antibody against CD33 in acute myeloid leukemia

    Journal: Blood Advances

    doi: 10.1182/bloodadvances.2017014373

    BC133 did not cross react with CD34 + CD38 – HSCs. (A) Cord blood mononuclear cells were purified using Ficoll-Paque density gradient centrifugation and were immunostained with anti-human CD3, CD19, CD38, CD34, and BC133 antibodies. To exclude T cells and B cells from analysis, cells were gated on CD3 – and CD19 – populations. Different populations of cells (labeled 1 to 6) were assessed for their binding to BC133. (B) HSCs and progenitor cells were isolated from cord blood mononuclear cells using Miltenyi CD34 Microbeads. (C) TDCC by ATC (E:T ratio, 10) in the presence of BC133 against the purified CD34 + cells and MOLM13 AML cells was tested by using chromium release assay.
    Figure Legend Snippet: BC133 did not cross react with CD34 + CD38 – HSCs. (A) Cord blood mononuclear cells were purified using Ficoll-Paque density gradient centrifugation and were immunostained with anti-human CD3, CD19, CD38, CD34, and BC133 antibodies. To exclude T cells and B cells from analysis, cells were gated on CD3 – and CD19 – populations. Different populations of cells (labeled 1 to 6) were assessed for their binding to BC133. (B) HSCs and progenitor cells were isolated from cord blood mononuclear cells using Miltenyi CD34 Microbeads. (C) TDCC by ATC (E:T ratio, 10) in the presence of BC133 against the purified CD34 + cells and MOLM13 AML cells was tested by using chromium release assay.

    Techniques Used: Purification, Gradient Centrifugation, Labeling, Binding Assay, Isolation, Release Assay

    2) Product Images from "Assessing the role of the T-box transcription factor Eomes in B cell differentiation during either Th1 or Th2 cell-biased responses"

    Article Title: Assessing the role of the T-box transcription factor Eomes in B cell differentiation during either Th1 or Th2 cell-biased responses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0208343

    Memory B cell formation in either Th1 or Th2 cell-biased responses proceeds in the absence of Eomes in B cells. Eomes f/f Cd23 cre/+ and littermate controls were immunized with NP-KLH precipitated in alum (A-C) or infected with influenza (D-E) to assess B cell memory formation. (A) Representative plots of antigen-specific memory B cells (NP + IgG1 + CD38 + B cells) in the spleen; representative of 5 mice at d14 and 5 mice at d28 post-immunization. (B) Summary plot of antigen-specific memory B cell frequency. (C) NP-binding IgG1-secreting B cells in the bone marrow was analyzed via ELISpot at d28 post-immunization; data is from 2 experiments, n = 5 per genotype. Error bars indicate mean ± SEM. No significance was detected using the Mann-Whitney non-parametric test. (D-E) HA + CD38 + IgG2c + B cells in the lung (D) or spleen (E) were assessed in influenza-infected mice 28 days post-infection. Data is from 2 experiments, n = 6–8 per genotype for splenic samples and n = 4–5 per genotype for lung samples. Error bars indicate mean ± SEM. No significance was detected using the Mann-Whitney non-parametric test.
    Figure Legend Snippet: Memory B cell formation in either Th1 or Th2 cell-biased responses proceeds in the absence of Eomes in B cells. Eomes f/f Cd23 cre/+ and littermate controls were immunized with NP-KLH precipitated in alum (A-C) or infected with influenza (D-E) to assess B cell memory formation. (A) Representative plots of antigen-specific memory B cells (NP + IgG1 + CD38 + B cells) in the spleen; representative of 5 mice at d14 and 5 mice at d28 post-immunization. (B) Summary plot of antigen-specific memory B cell frequency. (C) NP-binding IgG1-secreting B cells in the bone marrow was analyzed via ELISpot at d28 post-immunization; data is from 2 experiments, n = 5 per genotype. Error bars indicate mean ± SEM. No significance was detected using the Mann-Whitney non-parametric test. (D-E) HA + CD38 + IgG2c + B cells in the lung (D) or spleen (E) were assessed in influenza-infected mice 28 days post-infection. Data is from 2 experiments, n = 6–8 per genotype for splenic samples and n = 4–5 per genotype for lung samples. Error bars indicate mean ± SEM. No significance was detected using the Mann-Whitney non-parametric test.

    Techniques Used: Infection, Mouse Assay, Binding Assay, Enzyme-linked Immunospot, MANN-WHITNEY

    Eomes is not required for germinal center B cell formation or IgG2c isotype switching during influenza infection. Eomes f/f Cd23 cre/+ and littermate controls were infected with HKx31 influenza virus and mediastinal lymph node-derived and splenic B cells were analyzed 8 days post-infection. (A-B) Mature activated B cells (B220 + IgD lo ) were stained to identify germinal center B cells (CD95 hi CD38 lo ). (C-D) The frequency of germinal center B cells that had switched to IgG2c was assessed. (E-F) Frequency of the plasmablast population in either mediastinal lymph nodes (E) or spleen (F). (G-H) Total numbers of germinal center B cells (G) and plasmablasts (H) in the mediastinal lymph node. Data are pooled from 3 experiments, n = 8–10 per genotype. Error bars indicate mean ± SEM. No significant difference was detected using the Mann-Whitney non-parametric test.
    Figure Legend Snippet: Eomes is not required for germinal center B cell formation or IgG2c isotype switching during influenza infection. Eomes f/f Cd23 cre/+ and littermate controls were infected with HKx31 influenza virus and mediastinal lymph node-derived and splenic B cells were analyzed 8 days post-infection. (A-B) Mature activated B cells (B220 + IgD lo ) were stained to identify germinal center B cells (CD95 hi CD38 lo ). (C-D) The frequency of germinal center B cells that had switched to IgG2c was assessed. (E-F) Frequency of the plasmablast population in either mediastinal lymph nodes (E) or spleen (F). (G-H) Total numbers of germinal center B cells (G) and plasmablasts (H) in the mediastinal lymph node. Data are pooled from 3 experiments, n = 8–10 per genotype. Error bars indicate mean ± SEM. No significant difference was detected using the Mann-Whitney non-parametric test.

    Techniques Used: Infection, Derivative Assay, Staining, MANN-WHITNEY

    3) Product Images from "Patient-derived antibody recognizes a unique CD43 epitope expressed on all AML and has antileukemia activity in mice"

    Article Title: Patient-derived antibody recognizes a unique CD43 epitope expressed on all AML and has antileukemia activity in mice

    Journal: Blood Advances

    doi: 10.1182/bloodadvances.2017008342

    CD43s is overexpressed by myeloid malignancies. (A) AT1413 binding to CD34+ and CD38+ CD45dim AML blasts of patient 101. Bone marrow cells of this patient were isolated using a ficoll gradient and stored at diagnosis, precluding analysis of AT1413 interaction with nonmalignant granulocytes. (B) Representative examples of AT1413 binding to AML blasts obtained from newly diagnosed patients with AML or MDS . (C) AT1413 binding to extramedullary AML of 2 patients (myeloid sarcoma [chloroma] of inguinal node [1] and skin [2]). Paraffin-embedded THP-1 and Jurkat cells were used as a positive and negative control, respectively. Biotin immunoreactivity of antibody shown with streptavidin-HRP and the peroxidase substrate DAB. Scale bars, 20 μm. (D) Bone marrow of a patient with concomitant multiple myeloma and therapy-related AML. (Left) Hematoxylin and eosin staining. Asterisk, malignant double-nucleated plasma cell; arrowheads, AML blasts. Original magnification ×100. (Right) AT1413 staining of CD45dim AML blasts; CD138+ multiple myeloma plasma cells do not interact with AT1413. (E) AT1413 binding to CD45dim blasts of patients with AML, and to a lesser extent to CD45+ granulocytes and monocytes and absence of binding to CD45+ lymphocytes. The fold increase MFI of AT1413 compared with the negative control is indicated in gray (AT1002, filled gray histogram). Bone marrow (BL-079, BL-092, BL-095, BL-096, BL-099) or blood (BL-091, BL-106) of patients with AML was freshly obtained and red blood cells lysed before FACs analysis. RAEB, refractory anemia with excess blasts.
    Figure Legend Snippet: CD43s is overexpressed by myeloid malignancies. (A) AT1413 binding to CD34+ and CD38+ CD45dim AML blasts of patient 101. Bone marrow cells of this patient were isolated using a ficoll gradient and stored at diagnosis, precluding analysis of AT1413 interaction with nonmalignant granulocytes. (B) Representative examples of AT1413 binding to AML blasts obtained from newly diagnosed patients with AML or MDS . (C) AT1413 binding to extramedullary AML of 2 patients (myeloid sarcoma [chloroma] of inguinal node [1] and skin [2]). Paraffin-embedded THP-1 and Jurkat cells were used as a positive and negative control, respectively. Biotin immunoreactivity of antibody shown with streptavidin-HRP and the peroxidase substrate DAB. Scale bars, 20 μm. (D) Bone marrow of a patient with concomitant multiple myeloma and therapy-related AML. (Left) Hematoxylin and eosin staining. Asterisk, malignant double-nucleated plasma cell; arrowheads, AML blasts. Original magnification ×100. (Right) AT1413 staining of CD45dim AML blasts; CD138+ multiple myeloma plasma cells do not interact with AT1413. (E) AT1413 binding to CD45dim blasts of patients with AML, and to a lesser extent to CD45+ granulocytes and monocytes and absence of binding to CD45+ lymphocytes. The fold increase MFI of AT1413 compared with the negative control is indicated in gray (AT1002, filled gray histogram). Bone marrow (BL-079, BL-092, BL-095, BL-096, BL-099) or blood (BL-091, BL-106) of patients with AML was freshly obtained and red blood cells lysed before FACs analysis. RAEB, refractory anemia with excess blasts.

    Techniques Used: Binding Assay, Isolation, Negative Control, Staining, FACS

    4) Product Images from "B cell sub-types following acute malaria and associations with clinical immunity"

    Article Title: B cell sub-types following acute malaria and associations with clinical immunity

    Journal: Malaria Journal

    doi: 10.1186/s12936-016-1190-0

    Representative flow cytometry dot plot and gating hierarchy used to define B cell sub-types using cell markers CD3/14, CD19, CD10, CD38, CD27, and IgD. Using these markers, six CD19+B cell sub-sets are defined as: transitional (CD10 + CD27 − IgD + ), naïve (CD10 − CD27 − IgD + ), innate-like MBCs (CD10 − CD27 + IgD + ), classical MBCs (CD10 − CD27 + IgD − ), atypical MBCs (CD10 − CD27 − IgD − ), and plasmablasts/plasma cells (CD10 − CD27 hi CD38 hi IgD − )
    Figure Legend Snippet: Representative flow cytometry dot plot and gating hierarchy used to define B cell sub-types using cell markers CD3/14, CD19, CD10, CD38, CD27, and IgD. Using these markers, six CD19+B cell sub-sets are defined as: transitional (CD10 + CD27 − IgD + ), naïve (CD10 − CD27 − IgD + ), innate-like MBCs (CD10 − CD27 + IgD + ), classical MBCs (CD10 − CD27 + IgD − ), atypical MBCs (CD10 − CD27 − IgD − ), and plasmablasts/plasma cells (CD10 − CD27 hi CD38 hi IgD − )

    Techniques Used: Flow Cytometry, Cytometry

    5) Product Images from "OX40 is a potent immune stimulating target in late stage cancer patients"

    Article Title: OX40 is a potent immune stimulating target in late stage cancer patients

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-12-4174

    Anti-OX40 increases HLA-DR and CD38 expression on cycling CD8 + T cells (A) PBL gated on CD3 + CD8 + T cells and analyzed for Ki-67 from a patient in cohort 2, and CD3 + CD8 + Ki-67 + were assessed for expression of the activation markers HLA-DR and CD38 at different times after anti-OX40 administration. (B) Comparison of the mean percent of cycling CD3 + CD8 + that co-express HLA-DR and CD38 T cells in 11 anti-OX40 treated patients randomly selected from all three cohorts (black circles) and 9 normal donors (black triangles).
    Figure Legend Snippet: Anti-OX40 increases HLA-DR and CD38 expression on cycling CD8 + T cells (A) PBL gated on CD3 + CD8 + T cells and analyzed for Ki-67 from a patient in cohort 2, and CD3 + CD8 + Ki-67 + were assessed for expression of the activation markers HLA-DR and CD38 at different times after anti-OX40 administration. (B) Comparison of the mean percent of cycling CD3 + CD8 + that co-express HLA-DR and CD38 T cells in 11 anti-OX40 treated patients randomly selected from all three cohorts (black circles) and 9 normal donors (black triangles).

    Techniques Used: Expressing, Activation Assay

    6) Product Images from "Humanized Mice Engrafted With Human HSC Only or HSC and Thymus Support Comparable HIV-1 Replication, Immunopathology, and Responses to ART and Immune Therapy"

    Article Title: Humanized Mice Engrafted With Human HSC Only or HSC and Thymus Support Comparable HIV-1 Replication, Immunopathology, and Responses to ART and Immune Therapy

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00817

    IFNAR blockade reduces activation and PD-1 expression on CD8 T cells in both HIV-1 infected NRG-hu HSC and NRG-hu Thy/HSC mice under combined antiretroviral therapy (cART). NRG-hu HSC and NRG-hu Thy/HSC mice infected with HIV-1 were treated with cART from 4 to 12 weeks postinfection (wpi). From 7 to 10 wpi, the cART-treated mice were injected with α-IFNAR1 antibody or isotype control mIgG2a antibody. Mice were sacrificed at 12 wpi. (A,B) Summarized data show numbers of human CD4 and CD8 T cells from spleens of NRG-hu HSC (A) and NRG-hu Thy/HSC (B) mice. (C,D) Summarized data show percent HLA-DR + CD38 + of CD8 T cells from spleens of NRG-hu HSC (C) and NRG-hu Thy/HSC (D) mice. (E,F) Summarized data show percent PD-1 + of CD8 T cells from spleens of NRG-hu HSC (E) and NRG-hu Thy/HSC (F) mice. Shown are representative data from n = 3 (NRG-hu HSC/Mock), n = 3 (NRG-hu HSC/HIV-1), n = 4 (NRG-hu HSC/HIV-1/cART/mIgG2a), n = 4 (NRG-hu HSC/HIV-1/cART/α-IFNAR1), n = 4 (NRG-hu Thy/HSC/Mock), n = 4 (NRG-hu Thy/HSC/HIV-1), and n = 4 (NRG-hu Thy/HSC/HIV-1/cART/mIgG2a), n = 4 (NRG-hu Thy/HSC/HIV-1/cART/α-IFNAR1) mice. Each dot represents one individual mouse; bars indicate mean (* P
    Figure Legend Snippet: IFNAR blockade reduces activation and PD-1 expression on CD8 T cells in both HIV-1 infected NRG-hu HSC and NRG-hu Thy/HSC mice under combined antiretroviral therapy (cART). NRG-hu HSC and NRG-hu Thy/HSC mice infected with HIV-1 were treated with cART from 4 to 12 weeks postinfection (wpi). From 7 to 10 wpi, the cART-treated mice were injected with α-IFNAR1 antibody or isotype control mIgG2a antibody. Mice were sacrificed at 12 wpi. (A,B) Summarized data show numbers of human CD4 and CD8 T cells from spleens of NRG-hu HSC (A) and NRG-hu Thy/HSC (B) mice. (C,D) Summarized data show percent HLA-DR + CD38 + of CD8 T cells from spleens of NRG-hu HSC (C) and NRG-hu Thy/HSC (D) mice. (E,F) Summarized data show percent PD-1 + of CD8 T cells from spleens of NRG-hu HSC (E) and NRG-hu Thy/HSC (F) mice. Shown are representative data from n = 3 (NRG-hu HSC/Mock), n = 3 (NRG-hu HSC/HIV-1), n = 4 (NRG-hu HSC/HIV-1/cART/mIgG2a), n = 4 (NRG-hu HSC/HIV-1/cART/α-IFNAR1), n = 4 (NRG-hu Thy/HSC/Mock), n = 4 (NRG-hu Thy/HSC/HIV-1), and n = 4 (NRG-hu Thy/HSC/HIV-1/cART/mIgG2a), n = 4 (NRG-hu Thy/HSC/HIV-1/cART/α-IFNAR1) mice. Each dot represents one individual mouse; bars indicate mean (* P

    Techniques Used: Activation Assay, Expressing, Infection, Mouse Assay, Injection

    HIV-1-induced immunopathology in NRG-hu HSC and NRG-hu Thy/HSC mice. NRG-hu HSC and NRG-hu Thy/HSC mice were infected with HIV-1. Mice were sacrificed at 10 weeks postinfection. (A,B) Numbers of total human leukocytes, CD3 + T cells, CD4 T cells (CD3 + CD8 − ), and CD8 T cells (CD3 + CD8 − ) and in spleens of NRG-hu HSC (A) and NRG-hu Thy/HSC (B) mice. (C,D) Representative FACS plots and summarized data show the expression of CD38 and HLA-DR on CD8 T cells from spleen of NRG-hu HSC (C) and NRG-hu Thy/HSC (D) mice. (E,F) Representative FACS plots and summarized data show the expression of PD-1 on CD8 T cells from spleen of NRG-hu HSC (E) and NRG-hu Thy/HSC (F) mice. Shown are representative data from n = 3 (NRG-hu HSC/Mock), n = 5 (NRG-hu HSC/HIV-1), n = 3 (NRG-hu Thy/HSC/Mock), and n = 5 (NRG-hu Thy/HSC/HIV-1) mice reconstituted with HSCs/thymus from the same donor. Each dot represents one individual mouse; bars indicate mean (* P
    Figure Legend Snippet: HIV-1-induced immunopathology in NRG-hu HSC and NRG-hu Thy/HSC mice. NRG-hu HSC and NRG-hu Thy/HSC mice were infected with HIV-1. Mice were sacrificed at 10 weeks postinfection. (A,B) Numbers of total human leukocytes, CD3 + T cells, CD4 T cells (CD3 + CD8 − ), and CD8 T cells (CD3 + CD8 − ) and in spleens of NRG-hu HSC (A) and NRG-hu Thy/HSC (B) mice. (C,D) Representative FACS plots and summarized data show the expression of CD38 and HLA-DR on CD8 T cells from spleen of NRG-hu HSC (C) and NRG-hu Thy/HSC (D) mice. (E,F) Representative FACS plots and summarized data show the expression of PD-1 on CD8 T cells from spleen of NRG-hu HSC (E) and NRG-hu Thy/HSC (F) mice. Shown are representative data from n = 3 (NRG-hu HSC/Mock), n = 5 (NRG-hu HSC/HIV-1), n = 3 (NRG-hu Thy/HSC/Mock), and n = 5 (NRG-hu Thy/HSC/HIV-1) mice reconstituted with HSCs/thymus from the same donor. Each dot represents one individual mouse; bars indicate mean (* P

    Techniques Used: Mouse Assay, Infection, FACS, Expressing

    7) Product Images from "B cell–derived IL-6 initiates spontaneous germinal center formation during systemic autoimmunity"

    Article Title: B cell–derived IL-6 initiates spontaneous germinal center formation during systemic autoimmunity

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20170580

    IFN-γ synergizes with B cell activation signals to promote IL-6 production by mouse and human B cells. (A) Representative FACS plots showing CD45.1 + Was −/− versus CD45.2 + Was −/− Ifngr −/− B cells in GC and non-GC B cell compartments. (A, left) Gated on CD19 + B cells. (A, right) Gated on PNA + FAS + GC B cells (top) and PNA − FAS − non-GC B cells (bottom). Number equals percentage within gate. (B) Selection of CD45.1 + Was −/− versus CD45.2 + Was −/− Ifngr −/− B cells into the GC compartment. NS, not significant. (C) IL-6 from splenic WT (black), Tbx21 −/− (gray), and Ifngr −/− (white) B cells cultured for 48 h with anti–IgM, R848, anti–CD40 and/or IFN-γ. (D) IL-6 production by stimulated mouse B cells with or without ruxolitinib or tofacitinib (500 nM). (E) IL-6 in WT (black) and Stat1 −/− (white) B cells stimulated as indicated. (F) Surface PNA binding in WT (left) and Stat1 −/− (right) splenic B cells stimulated as indicated. (G) IL-6 intracellular staining at 72 h in cultured human B cells, gated as CD38 − CD27 − “naïve” (white) or CD38 + CD27 − “GC” (black) B cells. (H) IL-6 mean fluorescence intensity by intracellular staining in human CD38 + CD27 − B cells stimulated for 24 h. (I) IL-6 from human B cells stimulated for 72 h with or without ruxolitinib (500 nM). (B–E and G–I) Error bars indicate means ± SEM. *, P
    Figure Legend Snippet: IFN-γ synergizes with B cell activation signals to promote IL-6 production by mouse and human B cells. (A) Representative FACS plots showing CD45.1 + Was −/− versus CD45.2 + Was −/− Ifngr −/− B cells in GC and non-GC B cell compartments. (A, left) Gated on CD19 + B cells. (A, right) Gated on PNA + FAS + GC B cells (top) and PNA − FAS − non-GC B cells (bottom). Number equals percentage within gate. (B) Selection of CD45.1 + Was −/− versus CD45.2 + Was −/− Ifngr −/− B cells into the GC compartment. NS, not significant. (C) IL-6 from splenic WT (black), Tbx21 −/− (gray), and Ifngr −/− (white) B cells cultured for 48 h with anti–IgM, R848, anti–CD40 and/or IFN-γ. (D) IL-6 production by stimulated mouse B cells with or without ruxolitinib or tofacitinib (500 nM). (E) IL-6 in WT (black) and Stat1 −/− (white) B cells stimulated as indicated. (F) Surface PNA binding in WT (left) and Stat1 −/− (right) splenic B cells stimulated as indicated. (G) IL-6 intracellular staining at 72 h in cultured human B cells, gated as CD38 − CD27 − “naïve” (white) or CD38 + CD27 − “GC” (black) B cells. (H) IL-6 mean fluorescence intensity by intracellular staining in human CD38 + CD27 − B cells stimulated for 24 h. (I) IL-6 from human B cells stimulated for 72 h with or without ruxolitinib (500 nM). (B–E and G–I) Error bars indicate means ± SEM. *, P

    Techniques Used: Activation Assay, FACS, Selection, Cell Culture, Binding Assay, Staining, Fluorescence

    8) Product Images from "Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow"

    Article Title: Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16464-6

    Heterogeneity of switched memory B cells is differentially represented in spleen and bone marrow. Cells for single cell sequencing were FACSorted as IgG-expressing CD19 + CD38 + CD138 - GL7 − small lymphocytes. a Six transcriptionally defined clusters were identified by shared nearest neighbor (SNN) modularity optimization based clustering algorithm mapped to tSNE representation of spleen and bone marrow (BM) cells. tSNE coordinates and clustering was computed for 4754 from spleen and 2947 from BM cells, presentation is separated by organ. b Percentage of cells per cluster in each organ by mouse. c Distribution of IgG subclass mapped on tSNE. d Signature genes for each cluster, area under curve (AUC) of receiver-operator characteristics (ROC) of > 0.7. e Distribution of transcription levels for representative genes mapped on tSNE. Cells for single cell sequencing were FACSorted as IgG-expressing CD19 + CD38 + CD138 − GL7 − small lymphocytes from female C57BL/6 mice. Source data are provided as a Source Data file.
    Figure Legend Snippet: Heterogeneity of switched memory B cells is differentially represented in spleen and bone marrow. Cells for single cell sequencing were FACSorted as IgG-expressing CD19 + CD38 + CD138 - GL7 − small lymphocytes. a Six transcriptionally defined clusters were identified by shared nearest neighbor (SNN) modularity optimization based clustering algorithm mapped to tSNE representation of spleen and bone marrow (BM) cells. tSNE coordinates and clustering was computed for 4754 from spleen and 2947 from BM cells, presentation is separated by organ. b Percentage of cells per cluster in each organ by mouse. c Distribution of IgG subclass mapped on tSNE. d Signature genes for each cluster, area under curve (AUC) of receiver-operator characteristics (ROC) of > 0.7. e Distribution of transcription levels for representative genes mapped on tSNE. Cells for single cell sequencing were FACSorted as IgG-expressing CD19 + CD38 + CD138 − GL7 − small lymphocytes from female C57BL/6 mice. Source data are provided as a Source Data file.

    Techniques Used: Sequencing, Expressing, Mouse Assay

    The bone marrow contains a major population of isotype-switched non-proliferating memory B cells. a Quantification of NP-specific IgG2a/b + spleen, peripheral lymph nodes, blood and bone marrow (BM) memory B cells. Female C57BL/6 mice immunized with NP-KLH/LPS SC. Numbers of NP-binding IgG2b + cells in Spleen, BM, blood, and peripheral lymph nodes (pLN) determined by flow-cytometry on d421 or d426 post immunization; pooled from two independent experiments. OVA ctrl: staining controls from mice immunized with the irrelevant antigen ovalbumin (OVA). Gated for IgG2b + CD19 + CD38 + CD138 − GL7 − CD11c − IgM − IgD − PI − small lymphocytes (cf. Supplementary Fig. 5 ). Lines connect samples from one individual, paired one-sided t -test for spleen and BM samples. b Flow-cytometric quantification of Ki-67 expression in IgG2b + Bsm (IgG2b + CD19 + CD38 + CD138 − GL7 − CD11c − IgM − IgD − PI − small lymphocytes) splenic naïve (IgM + IgD + IgG2b − CD19 + CD38 + CD138 − GL7 − CD11c − PI − small lymphocytes) and germinal center (GC) (CD19 + CD38 lo GL7 + CD11c − PI − lymphocytes) B cells. Frequencies of Ki-67 + cells within the subset, data in right graph from two independent experiments using pooled cells from 4 to 20 C57BL/6 mice, paired one-sided t -test. c Flow-cytometric quantification of CD19 + B cells and IgG2b + memory B cells in female C57BL/6J mice treated with Cyclophosphamide (CyP) or untreated controls (PBS) after immunization with 3× NP-CGG/IFA. Analysis performed after 7 days of CyP. IgG2b + B cells quantified as IgG2b + CD19 + CD38 + CD138 − GL7 − CD11c − IgM − IgD − PI − small lymphocytes, CD19 + B cells as CD19 + CD138 − PI − lymphocytes (Welch’s test, one-sided). Representative data for one of two independent experiments ( n = 5 per group). Boxplot indicates median, first and third quartiles, whiskers: 1.5 IQR. d IgG2b + B memory cells (Ki-67 − IgD − Blimp1 − GFP − ) are dispersed as single cells throughout the BM. Arrows indicate IgG2b + DAPI + cells. Scale bar: 20 µm. Micrograph representative of five slides from two female C57BL/6 mice. e Co-localization of IgG2b + GFP − IgD − IgG2b + cells (arrows) with mesenchymal stromal cells. Arrows indicate IgG2b + DAPI + cells. Representative micrograph. Scale bars: 10 µm. f Co-localization of IgG2b + cells to mesenchymal stromal cells. Graph shows frequency of IgG2b + cells in direct contact (black) or within 10 μm (gray) of a cell stained for the respective molecule. g Flow cytometric quantification of surface expression of the VLA-4 and VLA-6 components CD29, CD49d, CD49f in spleen and BM IgG2b + Bsm. Gated for IgG2b + CD19 + CD38 + CD138 − GL7 − CD11c − IgM − IgD − PI − small lymphocytes, histogram plots representative of three biological replicates from C57BL/6 females. Source data for Fig. 1a–c, f are provided as a Source Data file.
    Figure Legend Snippet: The bone marrow contains a major population of isotype-switched non-proliferating memory B cells. a Quantification of NP-specific IgG2a/b + spleen, peripheral lymph nodes, blood and bone marrow (BM) memory B cells. Female C57BL/6 mice immunized with NP-KLH/LPS SC. Numbers of NP-binding IgG2b + cells in Spleen, BM, blood, and peripheral lymph nodes (pLN) determined by flow-cytometry on d421 or d426 post immunization; pooled from two independent experiments. OVA ctrl: staining controls from mice immunized with the irrelevant antigen ovalbumin (OVA). Gated for IgG2b + CD19 + CD38 + CD138 − GL7 − CD11c − IgM − IgD − PI − small lymphocytes (cf. Supplementary Fig. 5 ). Lines connect samples from one individual, paired one-sided t -test for spleen and BM samples. b Flow-cytometric quantification of Ki-67 expression in IgG2b + Bsm (IgG2b + CD19 + CD38 + CD138 − GL7 − CD11c − IgM − IgD − PI − small lymphocytes) splenic naïve (IgM + IgD + IgG2b − CD19 + CD38 + CD138 − GL7 − CD11c − PI − small lymphocytes) and germinal center (GC) (CD19 + CD38 lo GL7 + CD11c − PI − lymphocytes) B cells. Frequencies of Ki-67 + cells within the subset, data in right graph from two independent experiments using pooled cells from 4 to 20 C57BL/6 mice, paired one-sided t -test. c Flow-cytometric quantification of CD19 + B cells and IgG2b + memory B cells in female C57BL/6J mice treated with Cyclophosphamide (CyP) or untreated controls (PBS) after immunization with 3× NP-CGG/IFA. Analysis performed after 7 days of CyP. IgG2b + B cells quantified as IgG2b + CD19 + CD38 + CD138 − GL7 − CD11c − IgM − IgD − PI − small lymphocytes, CD19 + B cells as CD19 + CD138 − PI − lymphocytes (Welch’s test, one-sided). Representative data for one of two independent experiments ( n = 5 per group). Boxplot indicates median, first and third quartiles, whiskers: 1.5 IQR. d IgG2b + B memory cells (Ki-67 − IgD − Blimp1 − GFP − ) are dispersed as single cells throughout the BM. Arrows indicate IgG2b + DAPI + cells. Scale bar: 20 µm. Micrograph representative of five slides from two female C57BL/6 mice. e Co-localization of IgG2b + GFP − IgD − IgG2b + cells (arrows) with mesenchymal stromal cells. Arrows indicate IgG2b + DAPI + cells. Representative micrograph. Scale bars: 10 µm. f Co-localization of IgG2b + cells to mesenchymal stromal cells. Graph shows frequency of IgG2b + cells in direct contact (black) or within 10 μm (gray) of a cell stained for the respective molecule. g Flow cytometric quantification of surface expression of the VLA-4 and VLA-6 components CD29, CD49d, CD49f in spleen and BM IgG2b + Bsm. Gated for IgG2b + CD19 + CD38 + CD138 − GL7 − CD11c − IgM − IgD − PI − small lymphocytes, histogram plots representative of three biological replicates from C57BL/6 females. Source data for Fig. 1a–c, f are provided as a Source Data file.

    Techniques Used: Mouse Assay, Binding Assay, Flow Cytometry, Staining, Expressing, Immunofluorescence

    9) Product Images from "CD38 Is Robustly Induced in Human Macrophages and Monocytes in Inflammatory Conditions"

    Article Title: CD38 Is Robustly Induced in Human Macrophages and Monocytes in Inflammatory Conditions

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01593

    Increased CD38 expression in human M(LPS + IFN-γ) monocyte-derived macrophages (MDMs). Expression of human CD38 (A) , FPR2 (B) , GPR18 (C) , EGR2 (D) , and c-MYC (E) genes in human MDMs in unstimulated (M0), M(LPS + IFN-γ) (labeled M1 throughout figure), or M(IL-4) (labeled M2 throughout figure) human MDMs, as measured by real-time PCR. Gene expression level is expressed as fold change ± SD relative to M0 condition ( n = 7–8 independent samples, each generated from different donors; each independent sample value corresponds to the average of two technical replicates). One-way analysis of variance (ANOVA) with p values adjusted for multiple comparisons using Sidak’s multiple comparisons test, * p
    Figure Legend Snippet: Increased CD38 expression in human M(LPS + IFN-γ) monocyte-derived macrophages (MDMs). Expression of human CD38 (A) , FPR2 (B) , GPR18 (C) , EGR2 (D) , and c-MYC (E) genes in human MDMs in unstimulated (M0), M(LPS + IFN-γ) (labeled M1 throughout figure), or M(IL-4) (labeled M2 throughout figure) human MDMs, as measured by real-time PCR. Gene expression level is expressed as fold change ± SD relative to M0 condition ( n = 7–8 independent samples, each generated from different donors; each independent sample value corresponds to the average of two technical replicates). One-way analysis of variance (ANOVA) with p values adjusted for multiple comparisons using Sidak’s multiple comparisons test, * p

    Techniques Used: Expressing, Derivative Assay, Labeling, Real-time Polymerase Chain Reaction, Generated

    CD38 mRNA and protein expression is increased in human monocytic cell lines differentiated into M(LPS + IFN-γ) macrophages. Expression of CD38 and FPR2 mRNA in THP-1 (A,B) and U937-derived macrophages (C,D) in unstimulated (M0), M(LPS + IFN-γ)-stimulated (labeled M1 throughout figure), or M(IL-4)-stimulated (labeled M2 throughout figure) macrophages was measured by real-time PCR and expressed as mean relative expression ± SD ( n = 3 biological replicates with two technical replicates per sample). Gene expression is expressed as fold change ± SD relative to M0 condition. One-way analysis of variance with p values adjusted for multiple comparisons using Sidak’s multiple comparisons test compare M(LPS + IFN-γ) vs. M0 and M(LPS + IFN-γ) vs. M(IL-4). (E,F) Flow cytometry staining of surface FPR2 on x -axis, CD38 on y -axis in THP-1 (E) and U937 (F) cells. Flow plots correspond to total cells. Data shown are representative of n = 3 biological replicates. (G–J) Quantification of CD38 + cells and FPR2 + cells in THP-1 (G,H) or U937 cells (I,J) are expressed as percent of positive cells ± SD ( n = 3 biological replicates); ISO, isotype control; ** p
    Figure Legend Snippet: CD38 mRNA and protein expression is increased in human monocytic cell lines differentiated into M(LPS + IFN-γ) macrophages. Expression of CD38 and FPR2 mRNA in THP-1 (A,B) and U937-derived macrophages (C,D) in unstimulated (M0), M(LPS + IFN-γ)-stimulated (labeled M1 throughout figure), or M(IL-4)-stimulated (labeled M2 throughout figure) macrophages was measured by real-time PCR and expressed as mean relative expression ± SD ( n = 3 biological replicates with two technical replicates per sample). Gene expression is expressed as fold change ± SD relative to M0 condition. One-way analysis of variance with p values adjusted for multiple comparisons using Sidak’s multiple comparisons test compare M(LPS + IFN-γ) vs. M0 and M(LPS + IFN-γ) vs. M(IL-4). (E,F) Flow cytometry staining of surface FPR2 on x -axis, CD38 on y -axis in THP-1 (E) and U937 (F) cells. Flow plots correspond to total cells. Data shown are representative of n = 3 biological replicates. (G–J) Quantification of CD38 + cells and FPR2 + cells in THP-1 (G,H) or U937 cells (I,J) are expressed as percent of positive cells ± SD ( n = 3 biological replicates); ISO, isotype control; ** p

    Techniques Used: Expressing, Derivative Assay, Labeling, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Staining

    CD38 promotes inflammatory cytokine secretion in human macrophages. Human monocyte-derived macrophages (MDMs) were treated with 15 µM rhein or DMSO control (A) , 25 µM apigenin or DMSO (B) , or transfected with 100 µM CD38 siRNA cocktail or siRNA control (C) on day 5. On day 6, they were activated with LPS + IFN-γ for an additional 24 h prior to analysis. (A–C) IL-6 and IL-12p40 secretion was analyzed by ELISA from MDM supernatants. Graphs pool normalized (relative to corresponding experiment vehicle control or nonsense siRNA) data from three (A) , five (B) , or six (C) independent experiments/donors, with at least two technical replicates for each sample. Data are expressed as mean cytokine secretion ± SD relative to vehicle condition. (D) Quantification of CD38 + cells analyzed by flow cytometry from MDM transfected with control or CD38 siRNAs for data shown in panel (C) is expressed as percent of cells ± SD, n = 6 independent experiments/donors and two technical replicates per sample. (E) Quantification of IL-1β + MDMs analyzed by flow cytometry for data shown in panel (C) is expressed as percent of cells ± SD, n = 6 independent experiments/donors and two technical replicates per sample. (F) l -Lactate assays run using supernatants from panel (C) , n = 5 independent experiments/donors, with at least three technical replicates per sample. All data were analyzed by unpaired t tests. ** p
    Figure Legend Snippet: CD38 promotes inflammatory cytokine secretion in human macrophages. Human monocyte-derived macrophages (MDMs) were treated with 15 µM rhein or DMSO control (A) , 25 µM apigenin or DMSO (B) , or transfected with 100 µM CD38 siRNA cocktail or siRNA control (C) on day 5. On day 6, they were activated with LPS + IFN-γ for an additional 24 h prior to analysis. (A–C) IL-6 and IL-12p40 secretion was analyzed by ELISA from MDM supernatants. Graphs pool normalized (relative to corresponding experiment vehicle control or nonsense siRNA) data from three (A) , five (B) , or six (C) independent experiments/donors, with at least two technical replicates for each sample. Data are expressed as mean cytokine secretion ± SD relative to vehicle condition. (D) Quantification of CD38 + cells analyzed by flow cytometry from MDM transfected with control or CD38 siRNAs for data shown in panel (C) is expressed as percent of cells ± SD, n = 6 independent experiments/donors and two technical replicates per sample. (E) Quantification of IL-1β + MDMs analyzed by flow cytometry for data shown in panel (C) is expressed as percent of cells ± SD, n = 6 independent experiments/donors and two technical replicates per sample. (F) l -Lactate assays run using supernatants from panel (C) , n = 5 independent experiments/donors, with at least three technical replicates per sample. All data were analyzed by unpaired t tests. ** p

    Techniques Used: Derivative Assay, Transfection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    CD38 mean fluorescence intensity (MFI) in non-classical monocytes (NCMs) is elevated in active systemic lupus erythematosus (SLE). (A) Gating strategy: peripheral blood mononuclear cells isolated from healthy donors (HD) or SLE patients (panel 1) were gated for CD45 + cells (panel 2) prior to gating out CD66b − cells (panel 3) and CD3 − CD19 − CD56 − (panel 4) cells. The remaining population was analyzed with CD14 and CD16 after removing cells that are neither CD14 + nor CD16 + (NOT gate) (panel 5) to identify classical (CD14 ++ CD16 − ) (CM), intermediate (CD14 + CD16 + ) (IM) and non-classical (CD14 + CD16 + ) (NCM) monocytes (panel 6). A stricter gate that excluded CD14 − cells was also used for some analyses (box in panel 6 labeled CD14 low CD16 ++ ). (B) Example of how the relative percentage of CM, IM, and NCM among monocytes was calculated and how CM, IM, and NCM populations were analyzed for CD38 expression. ISO, isotype. (C) Percent of CD38 + cells within the CM, IM and NCM subsets in HD, inactive (SLE I = SLEDAI 0), or active (SLE A = SLEDAI > 4) patients. (D) A histogram of CD38 expression in NCM indicating how the CD38 hi subset was defined. A histogram from each group is overlaid, including an SLE I patient with an SLE Disease Activity Index (SLEDAI) of 0 and an SLE A patient with an SLEDAI of 16. (E) CD38 MFI within CD38 + CM, IM, and NCM populations of HD, SLE I , and SLE A patients. (F) Percent of CD38 hi cells within CM, IM, and NCM subsets in HD, SLE I , or SLE A patients. (G) CD38 MFI within CD38 + CD14 low CD16 ++ population in HD, SLE I , or SLE A patients. (H) Percent of CD38 hi cells within CD14 low CD16 ++ population in HD, SLE I , or SLE A patients. (C,E–H) Quantification of CD38 + cells (C) and CD38 hi cells (F,H) is expressed as percent of positive cells ± SD. Quantification of CD38 MFI (E,G) is expressed as MFI of positive cells ± SD. n = 9 donors for HD, n = 11 patients for SLE I , and n = 10 patients for SLE A . Each sample was run in duplicate, and duplicate values were averaged prior to analysis. One-way analysis of variance with p values adjusted for multiple comparisons using Tukey’s multiple comparisons test, * p
    Figure Legend Snippet: CD38 mean fluorescence intensity (MFI) in non-classical monocytes (NCMs) is elevated in active systemic lupus erythematosus (SLE). (A) Gating strategy: peripheral blood mononuclear cells isolated from healthy donors (HD) or SLE patients (panel 1) were gated for CD45 + cells (panel 2) prior to gating out CD66b − cells (panel 3) and CD3 − CD19 − CD56 − (panel 4) cells. The remaining population was analyzed with CD14 and CD16 after removing cells that are neither CD14 + nor CD16 + (NOT gate) (panel 5) to identify classical (CD14 ++ CD16 − ) (CM), intermediate (CD14 + CD16 + ) (IM) and non-classical (CD14 + CD16 + ) (NCM) monocytes (panel 6). A stricter gate that excluded CD14 − cells was also used for some analyses (box in panel 6 labeled CD14 low CD16 ++ ). (B) Example of how the relative percentage of CM, IM, and NCM among monocytes was calculated and how CM, IM, and NCM populations were analyzed for CD38 expression. ISO, isotype. (C) Percent of CD38 + cells within the CM, IM and NCM subsets in HD, inactive (SLE I = SLEDAI 0), or active (SLE A = SLEDAI > 4) patients. (D) A histogram of CD38 expression in NCM indicating how the CD38 hi subset was defined. A histogram from each group is overlaid, including an SLE I patient with an SLE Disease Activity Index (SLEDAI) of 0 and an SLE A patient with an SLEDAI of 16. (E) CD38 MFI within CD38 + CM, IM, and NCM populations of HD, SLE I , and SLE A patients. (F) Percent of CD38 hi cells within CM, IM, and NCM subsets in HD, SLE I , or SLE A patients. (G) CD38 MFI within CD38 + CD14 low CD16 ++ population in HD, SLE I , or SLE A patients. (H) Percent of CD38 hi cells within CD14 low CD16 ++ population in HD, SLE I , or SLE A patients. (C,E–H) Quantification of CD38 + cells (C) and CD38 hi cells (F,H) is expressed as percent of positive cells ± SD. Quantification of CD38 MFI (E,G) is expressed as MFI of positive cells ± SD. n = 9 donors for HD, n = 11 patients for SLE I , and n = 10 patients for SLE A . Each sample was run in duplicate, and duplicate values were averaged prior to analysis. One-way analysis of variance with p values adjusted for multiple comparisons using Tukey’s multiple comparisons test, * p

    Techniques Used: Fluorescence, Isolation, Labeling, Expressing, Activity Assay

    10) Product Images from "Severe fever with thrombocytopenia syndrome virus targets B cells in lethal human infections"

    Article Title: Severe fever with thrombocytopenia syndrome virus targets B cells in lethal human infections

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI129171

    Peripheral-blood plasmablasts do not show higher susceptibility to SFTSV infection among peripheral-blood B cells in healthy adults. ( A ) Flow cytometry gating strategy to define lymphocyte subsets by CD3, CD19, CD27, and CD38 in SFTSV-inoculated PBMCs obtained from healthy donors ( n = 11). The infectivity of SFTSV in each subset was determined by intracellular staining with DyLight 488–conjugated anti–SFTSV N antibody at 24 hours after inoculation. ( B ) Comparison of SFTSV infectivity among CD19 − (blue), CD19 + (red), or CD3 + (green) lymphocytes in SFTSV-inoculated PBMCs. CD19 + B cells showed higher susceptibility to SFTSV than CD3 + T cells or CD19 − non-B cells. Scatter plots also show mean ± SD. **** P
    Figure Legend Snippet: Peripheral-blood plasmablasts do not show higher susceptibility to SFTSV infection among peripheral-blood B cells in healthy adults. ( A ) Flow cytometry gating strategy to define lymphocyte subsets by CD3, CD19, CD27, and CD38 in SFTSV-inoculated PBMCs obtained from healthy donors ( n = 11). The infectivity of SFTSV in each subset was determined by intracellular staining with DyLight 488–conjugated anti–SFTSV N antibody at 24 hours after inoculation. ( B ) Comparison of SFTSV infectivity among CD19 − (blue), CD19 + (red), or CD3 + (green) lymphocytes in SFTSV-inoculated PBMCs. CD19 + B cells showed higher susceptibility to SFTSV than CD3 + T cells or CD19 − non-B cells. Scatter plots also show mean ± SD. **** P

    Techniques Used: Infection, Flow Cytometry, Staining

    Class-switched IgG + B cells are more susceptible to SFTSV infection than class-unswitched IgM + B cells in lymphoid and nonlymphoid organs. ( A ) Representative images of chromogenic IHC staining for SFTSV N protein (left panel) or multiplex IHC staining for immunoglobulin heavy chains (IgA, brown; IgM, purple; IgG, green; right panel) on serial tissue sections of lymph node from an individual with fatal SFTS. The arrowhead in the right panel indicates an IgM + cell. Scale bars: 50 μm. ( B ) Representative images of chromogenic multiplex IHC staining for SFTSV (green) with activated lymphocyte marker (CD38, brown; left panel); plasmablast-lineage marker (MUM1, brown; middle panel); and IgG (brown; right panel). Arrowheads indicate colocalization of staining in cells. Scale bars: 10 μm. ( C ) Comparison between immunoglobulin heavy chain ( IGH ) mRNA levels ( IGHG and IGHM ) and SFTSV viral RNA levels in tissue sections of lymph nodes ( n = 58, left panel) and nonlymphoid organs ( n = 36, right panel) including the liver, adrenal gland, kidney, heart, and bladder, quantified by real-time RT-PCR. The SFTSV RNA load was positively correlated with the IGHG mRNA level in lymph node (Spearman’s coefficient = 0.6027, P
    Figure Legend Snippet: Class-switched IgG + B cells are more susceptible to SFTSV infection than class-unswitched IgM + B cells in lymphoid and nonlymphoid organs. ( A ) Representative images of chromogenic IHC staining for SFTSV N protein (left panel) or multiplex IHC staining for immunoglobulin heavy chains (IgA, brown; IgM, purple; IgG, green; right panel) on serial tissue sections of lymph node from an individual with fatal SFTS. The arrowhead in the right panel indicates an IgM + cell. Scale bars: 50 μm. ( B ) Representative images of chromogenic multiplex IHC staining for SFTSV (green) with activated lymphocyte marker (CD38, brown; left panel); plasmablast-lineage marker (MUM1, brown; middle panel); and IgG (brown; right panel). Arrowheads indicate colocalization of staining in cells. Scale bars: 10 μm. ( C ) Comparison between immunoglobulin heavy chain ( IGH ) mRNA levels ( IGHG and IGHM ) and SFTSV viral RNA levels in tissue sections of lymph nodes ( n = 58, left panel) and nonlymphoid organs ( n = 36, right panel) including the liver, adrenal gland, kidney, heart, and bladder, quantified by real-time RT-PCR. The SFTSV RNA load was positively correlated with the IGHG mRNA level in lymph node (Spearman’s coefficient = 0.6027, P

    Techniques Used: Infection, Immunohistochemistry, Staining, Multiplex Assay, Marker, Quantitative RT-PCR

    11) Product Images from "Potent neutralizing monoclonal antibodies against Ebola virus isolated from vaccinated donors"

    Article Title: Potent neutralizing monoclonal antibodies against Ebola virus isolated from vaccinated donors

    Journal: mAbs

    doi: 10.1080/19420862.2020.1742457

    Isolation of GP-specific monoclonal antibodies. (a) Binding capacity of the serum of vaccine-immunized subjects # 024, 057, and 088 to EBOV GP, BDBV GP, and SUDV GP. Values represent the difference in optical density (OD) between sera (1:10,000) on day 28 post-boost immunization and day 0 from the same donor. See also Figure S1a. (b) Neutralizing capacity of the serum of vaccine-immunized subjects # 024, 057, and 088 against pseudotyped HIV-EBOV GP-Luc. Data on the curve represent the difference in neutralization ability between sera on day 28 post-boost immunization and day 0 from the same donor. (c) Sorting of CD3 − /CD38 − /IgG + /CD19 + /CD27 + /GP∆Muc + single memory B cells obtained from PBMCs one month post-boost immunization to identify GP-specific mAbs. (d) Number of specific or cross-reactive antibodies identified using the supernatants of Ig genes linear expression cassettes. See also Figure S1b. (e) Correlation between GP sequence similarity to EBOV GP and number of binding antibodies. (f) Number of antibodies binding to different truncated EBOV GPs determined by ELISA using 293 T supernatants. See also Figure S1b.
    Figure Legend Snippet: Isolation of GP-specific monoclonal antibodies. (a) Binding capacity of the serum of vaccine-immunized subjects # 024, 057, and 088 to EBOV GP, BDBV GP, and SUDV GP. Values represent the difference in optical density (OD) between sera (1:10,000) on day 28 post-boost immunization and day 0 from the same donor. See also Figure S1a. (b) Neutralizing capacity of the serum of vaccine-immunized subjects # 024, 057, and 088 against pseudotyped HIV-EBOV GP-Luc. Data on the curve represent the difference in neutralization ability between sera on day 28 post-boost immunization and day 0 from the same donor. (c) Sorting of CD3 − /CD38 − /IgG + /CD19 + /CD27 + /GP∆Muc + single memory B cells obtained from PBMCs one month post-boost immunization to identify GP-specific mAbs. (d) Number of specific or cross-reactive antibodies identified using the supernatants of Ig genes linear expression cassettes. See also Figure S1b. (e) Correlation between GP sequence similarity to EBOV GP and number of binding antibodies. (f) Number of antibodies binding to different truncated EBOV GPs determined by ELISA using 293 T supernatants. See also Figure S1b.

    Techniques Used: Isolation, Binding Assay, Neutralization, Expressing, Sequencing, Enzyme-linked Immunosorbent Assay

    12) Product Images from "A role for gut-associated lymphoid tissue in shaping the human B cell repertoire"

    Article Title: A role for gut-associated lymphoid tissue in shaping the human B cell repertoire

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20122465

    Expression of β7 integrin on TS B cells is impaired in SLE. (A) PBMCs isolated from healthy donors and patients with SLE were analyzed by flow cytometry for the expression of CD19, CD20, CD27, CD24, CD38, and β7. Graph displays the percentages of β7 + healthy or SLE T1, T2, and mature B cells. ***, P
    Figure Legend Snippet: Expression of β7 integrin on TS B cells is impaired in SLE. (A) PBMCs isolated from healthy donors and patients with SLE were analyzed by flow cytometry for the expression of CD19, CD20, CD27, CD24, CD38, and β7. Graph displays the percentages of β7 + healthy or SLE T1, T2, and mature B cells. ***, P

    Techniques Used: Expressing, Isolation, Flow Cytometry, Cytometry

    A subpopulation of transitional B cells is recruited to the GALT. (A and B) Healthy peripheral blood and Peyer’s patch B cells were analyzed by flow cytometry for the expression of CD19, CD20, CD27, CD24, CD38, CD10, and IgD. FACS plots display the percentages of T1, T2, and naive B cells, and histograms display CD10 and IgD expression for each subset, healthy control blood (A) and Peyer’s patches (B). Plots show one of four experiments with similar results. (C) Confocal microscopy of human Peyer’s patch. CD10 + (red), IgD + (green) transitional B cells are highlighted by arrows at the periphery of the germinal center. GC, germinal center; MZ, marginal zone. One of three experiments with similar results is shown. (D) Immunohistochemistry of GALT identifies CD10 + (pink) and IgD + (brown) transitional B cells in the periphery of the germinal center. One of three experiments with similar results is shown. (E) Immunohistochemistry of GALT showing CD10 + cells (brown) scattered in the mantle zone of GALT. One of three experiments with similar results is shown. (F and G) T1, T2, and naive B cells were identified by flow cytometry (as in A and B) in F. Perfusates from normal liver grafts and matched donor blood (G) are shown. One of three experiments with similar results is shown. (H) Ratios of the %T2 to %T1 subpopulations in normal blood, matched liver donor blood, liver graft perfusates, and normal Peyer’s patches. Error bars show mean ± SEM. Statistical test: one-way ANOVA. n = up to 15. ***, P
    Figure Legend Snippet: A subpopulation of transitional B cells is recruited to the GALT. (A and B) Healthy peripheral blood and Peyer’s patch B cells were analyzed by flow cytometry for the expression of CD19, CD20, CD27, CD24, CD38, CD10, and IgD. FACS plots display the percentages of T1, T2, and naive B cells, and histograms display CD10 and IgD expression for each subset, healthy control blood (A) and Peyer’s patches (B). Plots show one of four experiments with similar results. (C) Confocal microscopy of human Peyer’s patch. CD10 + (red), IgD + (green) transitional B cells are highlighted by arrows at the periphery of the germinal center. GC, germinal center; MZ, marginal zone. One of three experiments with similar results is shown. (D) Immunohistochemistry of GALT identifies CD10 + (pink) and IgD + (brown) transitional B cells in the periphery of the germinal center. One of three experiments with similar results is shown. (E) Immunohistochemistry of GALT showing CD10 + cells (brown) scattered in the mantle zone of GALT. One of three experiments with similar results is shown. (F and G) T1, T2, and naive B cells were identified by flow cytometry (as in A and B) in F. Perfusates from normal liver grafts and matched donor blood (G) are shown. One of three experiments with similar results is shown. (H) Ratios of the %T2 to %T1 subpopulations in normal blood, matched liver donor blood, liver graft perfusates, and normal Peyer’s patches. Error bars show mean ± SEM. Statistical test: one-way ANOVA. n = up to 15. ***, P

    Techniques Used: Flow Cytometry, Cytometry, Expressing, FACS, Confocal Microscopy, Immunohistochemistry

    GALT transitional B cells are activated in vivo and in vitro in response to intestinal bacteria. (A) Mononuclear cells were isolated from Peyer’s patches and analyzed directly ex vivo by flow cytometry for their expression of CD19, CD10, IgD, and phospho-BTK, phospho-Syk, and phospho-ERK. Representative histograms displaying the degree of BTK, Syk, and ERK phosphorylation for CD19 + CD10 + IgD + transitional (TS) and CD19 + CD10 + IgD − germinal center (GC) B cells. Shown is one of three experiments with similar results. (B) PBMCs were stimulated polyclonally for 30 min with CpG, anti-IgM, and heat-inactivated intestinal bacteria as a positive control for B cell kinase phosphorylation, or left unstimulated. After stimulation, cells were analyzed by flow cytometry for the expression of CD19, CD20, CD27, CD24, CD38, and phospho-BTK, phospho-Syk, and phospho-ERK. Histograms display the degree of BTK, Syk, and ERK phosphorylation for T1, T2, and naive B cells. Histograms show one of three experiments. (C) FACS-sorted T1 cells (CD19 + CD20 + CD27 − CD24 ++ CD38 ++ ), T2 cells (CD19 + CD20 + CD27 − CD24 + CD38 + ), and naive mature B cells (CD19 + CD20 + CD27 − CD24 − CD38 − ) isolated from normal blood were incubated with combinations of heat-inactivated intestinal bacteria, CpG, and anti-IgM for 30 min, followed by intracellular staining for phospho-BTK. Mean values + SD from three independent experiments are shows. (D and E) Autoantibody secretion by in vitro stimulated peripheral blood and Peyer’s patch B cells. T1, T2, and naive mature B cells were FACS sorted (sort strategy as in Fig. S2 A ) from normal blood and Peyer’s patches (one experiment with pooled cells from three donors) and were incubated with heat-inactivated intestinal bacteria for 8 d. Supernatants were screened for secreted autoantibodies by Hep-2 ELISA. Graphs show Hep-2 binding IgG (D) and IgA (E) autoantibodies. Data are standardized so that reactivity of supernatant from blood naive cells is 1 for each isotype. (F and G) IGHV genes were amplified by PCR from FACS-sorted TS and GC B cells isolated from Peyer’s patches of seven different donors (gated and sorted as shown in Fig. S2 B ) from two sorts. (F) Pooled data of the number of mutations in GC or TS IGHV genes. Mann-Whitney test, P = 0.0001. (G) Number of mutations in GC or TS IGHV genes segregated by donor. Horizontal bars show the mean number of mutations. (H and I) An example of mutations in clonally related germinal center B cells. (H) Examples of IGHV sequencing of germinal center B cells showing four clonally related sequences deriving from a common ancestor. (I) Graphical explanation of the relation of the clones in H to a common ancestor and their germline precursor.
    Figure Legend Snippet: GALT transitional B cells are activated in vivo and in vitro in response to intestinal bacteria. (A) Mononuclear cells were isolated from Peyer’s patches and analyzed directly ex vivo by flow cytometry for their expression of CD19, CD10, IgD, and phospho-BTK, phospho-Syk, and phospho-ERK. Representative histograms displaying the degree of BTK, Syk, and ERK phosphorylation for CD19 + CD10 + IgD + transitional (TS) and CD19 + CD10 + IgD − germinal center (GC) B cells. Shown is one of three experiments with similar results. (B) PBMCs were stimulated polyclonally for 30 min with CpG, anti-IgM, and heat-inactivated intestinal bacteria as a positive control for B cell kinase phosphorylation, or left unstimulated. After stimulation, cells were analyzed by flow cytometry for the expression of CD19, CD20, CD27, CD24, CD38, and phospho-BTK, phospho-Syk, and phospho-ERK. Histograms display the degree of BTK, Syk, and ERK phosphorylation for T1, T2, and naive B cells. Histograms show one of three experiments. (C) FACS-sorted T1 cells (CD19 + CD20 + CD27 − CD24 ++ CD38 ++ ), T2 cells (CD19 + CD20 + CD27 − CD24 + CD38 + ), and naive mature B cells (CD19 + CD20 + CD27 − CD24 − CD38 − ) isolated from normal blood were incubated with combinations of heat-inactivated intestinal bacteria, CpG, and anti-IgM for 30 min, followed by intracellular staining for phospho-BTK. Mean values + SD from three independent experiments are shows. (D and E) Autoantibody secretion by in vitro stimulated peripheral blood and Peyer’s patch B cells. T1, T2, and naive mature B cells were FACS sorted (sort strategy as in Fig. S2 A ) from normal blood and Peyer’s patches (one experiment with pooled cells from three donors) and were incubated with heat-inactivated intestinal bacteria for 8 d. Supernatants were screened for secreted autoantibodies by Hep-2 ELISA. Graphs show Hep-2 binding IgG (D) and IgA (E) autoantibodies. Data are standardized so that reactivity of supernatant from blood naive cells is 1 for each isotype. (F and G) IGHV genes were amplified by PCR from FACS-sorted TS and GC B cells isolated from Peyer’s patches of seven different donors (gated and sorted as shown in Fig. S2 B ) from two sorts. (F) Pooled data of the number of mutations in GC or TS IGHV genes. Mann-Whitney test, P = 0.0001. (G) Number of mutations in GC or TS IGHV genes segregated by donor. Horizontal bars show the mean number of mutations. (H and I) An example of mutations in clonally related germinal center B cells. (H) Examples of IGHV sequencing of germinal center B cells showing four clonally related sequences deriving from a common ancestor. (I) Graphical explanation of the relation of the clones in H to a common ancestor and their germline precursor.

    Techniques Used: In Vivo, In Vitro, Isolation, Ex Vivo, Flow Cytometry, Cytometry, Expressing, Positive Control, FACS, Incubation, Staining, Enzyme-linked Immunosorbent Assay, Binding Assay, Amplification, Polymerase Chain Reaction, MANN-WHITNEY, Sequencing, Clone Assay

    13) Product Images from "FOXP3+Helios+ regulatory T cells, immune activation and advancing disease in HIV infected children"

    Article Title: FOXP3+Helios+ regulatory T cells, immune activation and advancing disease in HIV infected children

    Journal: Journal of acquired immune deficiency syndromes (1999)

    doi: 10.1097/QAI.0000000000001000

    CD38+, Ki67+, and PD-1+ memory CD4 T cells correlate with frequency and phenotype of memory FOXP+Helios+ Tregs in HIV infected children
    Figure Legend Snippet: CD38+, Ki67+, and PD-1+ memory CD4 T cells correlate with frequency and phenotype of memory FOXP+Helios+ Tregs in HIV infected children

    Techniques Used: Infection

    Elevated CD38, Ki67, and PD-1 expression in memory FOXP3-Helios- CD4 T cells and FOXP3+Helios+ Tregs of HIV+ children
    Figure Legend Snippet: Elevated CD38, Ki67, and PD-1 expression in memory FOXP3-Helios- CD4 T cells and FOXP3+Helios+ Tregs of HIV+ children

    Techniques Used: Expressing

    14) Product Images from "Humanized Mice Engrafted With Human HSC Only or HSC and Thymus Support Comparable HIV-1 Replication, Immunopathology, and Responses to ART and Immune Therapy"

    Article Title: Humanized Mice Engrafted With Human HSC Only or HSC and Thymus Support Comparable HIV-1 Replication, Immunopathology, and Responses to ART and Immune Therapy

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00817

    IFNAR blockade reduces activation and PD-1 expression on CD8 T cells in both HIV-1 infected NRG-hu HSC and NRG-hu Thy/HSC mice under combined antiretroviral therapy (cART). NRG-hu HSC and NRG-hu Thy/HSC mice infected with HIV-1 were treated with cART from 4 to 12 weeks postinfection (wpi). From 7 to 10 wpi, the cART-treated mice were injected with α-IFNAR1 antibody or isotype control mIgG2a antibody. Mice were sacrificed at 12 wpi. (A,B) Summarized data show numbers of human CD4 and CD8 T cells from spleens of NRG-hu HSC (A) and NRG-hu Thy/HSC (B) mice. (C,D) Summarized data show percent HLA-DR + CD38 + of CD8 T cells from spleens of NRG-hu HSC (C) and NRG-hu Thy/HSC (D) mice. (E,F) Summarized data show percent PD-1 + of CD8 T cells from spleens of NRG-hu HSC (E) and NRG-hu Thy/HSC (F) mice. Shown are representative data from n = 3 (NRG-hu HSC/Mock), n = 3 (NRG-hu HSC/HIV-1), n = 4 (NRG-hu HSC/HIV-1/cART/mIgG2a), n = 4 (NRG-hu HSC/HIV-1/cART/α-IFNAR1), n = 4 (NRG-hu Thy/HSC/Mock), n = 4 (NRG-hu Thy/HSC/HIV-1), and n = 4 (NRG-hu Thy/HSC/HIV-1/cART/mIgG2a), n = 4 (NRG-hu Thy/HSC/HIV-1/cART/α-IFNAR1) mice. Each dot represents one individual mouse; bars indicate mean (* P
    Figure Legend Snippet: IFNAR blockade reduces activation and PD-1 expression on CD8 T cells in both HIV-1 infected NRG-hu HSC and NRG-hu Thy/HSC mice under combined antiretroviral therapy (cART). NRG-hu HSC and NRG-hu Thy/HSC mice infected with HIV-1 were treated with cART from 4 to 12 weeks postinfection (wpi). From 7 to 10 wpi, the cART-treated mice were injected with α-IFNAR1 antibody or isotype control mIgG2a antibody. Mice were sacrificed at 12 wpi. (A,B) Summarized data show numbers of human CD4 and CD8 T cells from spleens of NRG-hu HSC (A) and NRG-hu Thy/HSC (B) mice. (C,D) Summarized data show percent HLA-DR + CD38 + of CD8 T cells from spleens of NRG-hu HSC (C) and NRG-hu Thy/HSC (D) mice. (E,F) Summarized data show percent PD-1 + of CD8 T cells from spleens of NRG-hu HSC (E) and NRG-hu Thy/HSC (F) mice. Shown are representative data from n = 3 (NRG-hu HSC/Mock), n = 3 (NRG-hu HSC/HIV-1), n = 4 (NRG-hu HSC/HIV-1/cART/mIgG2a), n = 4 (NRG-hu HSC/HIV-1/cART/α-IFNAR1), n = 4 (NRG-hu Thy/HSC/Mock), n = 4 (NRG-hu Thy/HSC/HIV-1), and n = 4 (NRG-hu Thy/HSC/HIV-1/cART/mIgG2a), n = 4 (NRG-hu Thy/HSC/HIV-1/cART/α-IFNAR1) mice. Each dot represents one individual mouse; bars indicate mean (* P

    Techniques Used: Activation Assay, Expressing, Infection, Mouse Assay, Injection

    HIV-1-induced immunopathology in NRG-hu HSC and NRG-hu Thy/HSC mice. NRG-hu HSC and NRG-hu Thy/HSC mice were infected with HIV-1. Mice were sacrificed at 10 weeks postinfection. (A,B) Numbers of total human leukocytes, CD3 + T cells, CD4 T cells (CD3 + CD8 − ), and CD8 T cells (CD3 + CD8 − ) and in spleens of NRG-hu HSC (A) and NRG-hu Thy/HSC (B) mice. (C,D) Representative FACS plots and summarized data show the expression of CD38 and HLA-DR on CD8 T cells from spleen of NRG-hu HSC (C) and NRG-hu Thy/HSC (D) mice. (E,F) Representative FACS plots and summarized data show the expression of PD-1 on CD8 T cells from spleen of NRG-hu HSC (E) and NRG-hu Thy/HSC (F) mice. Shown are representative data from n = 3 (NRG-hu HSC/Mock), n = 5 (NRG-hu HSC/HIV-1), n = 3 (NRG-hu Thy/HSC/Mock), and n = 5 (NRG-hu Thy/HSC/HIV-1) mice reconstituted with HSCs/thymus from the same donor. Each dot represents one individual mouse; bars indicate mean (* P
    Figure Legend Snippet: HIV-1-induced immunopathology in NRG-hu HSC and NRG-hu Thy/HSC mice. NRG-hu HSC and NRG-hu Thy/HSC mice were infected with HIV-1. Mice were sacrificed at 10 weeks postinfection. (A,B) Numbers of total human leukocytes, CD3 + T cells, CD4 T cells (CD3 + CD8 − ), and CD8 T cells (CD3 + CD8 − ) and in spleens of NRG-hu HSC (A) and NRG-hu Thy/HSC (B) mice. (C,D) Representative FACS plots and summarized data show the expression of CD38 and HLA-DR on CD8 T cells from spleen of NRG-hu HSC (C) and NRG-hu Thy/HSC (D) mice. (E,F) Representative FACS plots and summarized data show the expression of PD-1 on CD8 T cells from spleen of NRG-hu HSC (E) and NRG-hu Thy/HSC (F) mice. Shown are representative data from n = 3 (NRG-hu HSC/Mock), n = 5 (NRG-hu HSC/HIV-1), n = 3 (NRG-hu Thy/HSC/Mock), and n = 5 (NRG-hu Thy/HSC/HIV-1) mice reconstituted with HSCs/thymus from the same donor. Each dot represents one individual mouse; bars indicate mean (* P

    Techniques Used: Mouse Assay, Infection, FACS, Expressing

    15) Product Images from "Immune activation despite preserved CD4 T cells in perinatally HIV-infected children and adolescents"

    Article Title: Immune activation despite preserved CD4 T cells in perinatally HIV-infected children and adolescents

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190332

    Antiretroviral therapy lowers immune activation rapidly and persistently. Comparisons of (A) the CD4 percentages and (B-E) frequencies of the following IA markers in paired longitudinal samples pre-ART (T0), 5–7 months post-ART (T1), and 10–16 months post-ART (T2): CD38+HLA-DR+ (B) CD8 and (C) CD4 T cells and (D) CD38+ (E) and Ki67+ memory CD4 T cells. Right graphs show comparison between IA markers in HIV- and the prospective cohort pre- and post-ART. Bars represent median values with IQRs. P values were calculated using the paired Wilcoxon matched-pairs signed rank test (left graphs) and the Kruskal-Wallis test corrected for multiple comparisons by controlling the false discovery rate with the Benjamini, Krieger, and Yekutieli test (right graphs). **** p
    Figure Legend Snippet: Antiretroviral therapy lowers immune activation rapidly and persistently. Comparisons of (A) the CD4 percentages and (B-E) frequencies of the following IA markers in paired longitudinal samples pre-ART (T0), 5–7 months post-ART (T1), and 10–16 months post-ART (T2): CD38+HLA-DR+ (B) CD8 and (C) CD4 T cells and (D) CD38+ (E) and Ki67+ memory CD4 T cells. Right graphs show comparison between IA markers in HIV- and the prospective cohort pre- and post-ART. Bars represent median values with IQRs. P values were calculated using the paired Wilcoxon matched-pairs signed rank test (left graphs) and the Kruskal-Wallis test corrected for multiple comparisons by controlling the false discovery rate with the Benjamini, Krieger, and Yekutieli test (right graphs). **** p

    Techniques Used: Activation Assay, IA

    Significant CD4 T cell activation in ART-CD4 hi children and adolescents. (A) Comparison of frequencies of the CD38+HLA-DR+ CD4 T cells in HIV-, ART-CD4 hi , ART-CD4 lo and ART+ children and adolescents. Percentages of (B) CD38+ and (C) Ki67+ cells within in memory CD4 T cells in HIV-, ART-CD4 hi , ART-CD4 lo and ART+ children and adolescents. To identify memory populations, CD4 T cell were first gated on CD45RO+ CD4 T cells. Bars represent median values with IQRs. P values were calculated using the Kruskal-Wallis test corrected for multiple comparisons by controlling the false discovery rate with the Benjamini, Krieger, and Yekutieli test. **** p
    Figure Legend Snippet: Significant CD4 T cell activation in ART-CD4 hi children and adolescents. (A) Comparison of frequencies of the CD38+HLA-DR+ CD4 T cells in HIV-, ART-CD4 hi , ART-CD4 lo and ART+ children and adolescents. Percentages of (B) CD38+ and (C) Ki67+ cells within in memory CD4 T cells in HIV-, ART-CD4 hi , ART-CD4 lo and ART+ children and adolescents. To identify memory populations, CD4 T cell were first gated on CD45RO+ CD4 T cells. Bars represent median values with IQRs. P values were calculated using the Kruskal-Wallis test corrected for multiple comparisons by controlling the false discovery rate with the Benjamini, Krieger, and Yekutieli test. **** p

    Techniques Used: Activation Assay

    HIV disease progression in ART-CD4 hi children and adolescents. Comparison of the (A) CD4 percent and (B) HIV viral load in HIV-, ART-CD4 hi , ART- and ART+ children and adolescents. (C) Comparison of the %CD38+DR+ CD8 T cells in HIV-, ART-CD4 hi , ART-CD4 lo and ART+ children and adolescents. Bars represent median values with IQRs. P values were calculated using the Kruskal-Wallis test corrected for multiple comparisons by controlling the false discovery rate with the Benjamini, Krieger, and Yekutieli test. **** p
    Figure Legend Snippet: HIV disease progression in ART-CD4 hi children and adolescents. Comparison of the (A) CD4 percent and (B) HIV viral load in HIV-, ART-CD4 hi , ART- and ART+ children and adolescents. (C) Comparison of the %CD38+DR+ CD8 T cells in HIV-, ART-CD4 hi , ART-CD4 lo and ART+ children and adolescents. Bars represent median values with IQRs. P values were calculated using the Kruskal-Wallis test corrected for multiple comparisons by controlling the false discovery rate with the Benjamini, Krieger, and Yekutieli test. **** p

    Techniques Used:

    16) Product Images from "CD38 Is Robustly Induced in Human Macrophages and Monocytes in Inflammatory Conditions"

    Article Title: CD38 Is Robustly Induced in Human Macrophages and Monocytes in Inflammatory Conditions

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01593

    Increased CD38 expression in human M(LPS + IFN-γ) monocyte-derived macrophages (MDMs). Expression of human CD38 (A) , FPR2 (B) , GPR18 (C) , EGR2 (D) , and c-MYC (E) genes in human MDMs in unstimulated (M0), M(LPS + IFN-γ) (labeled M1 throughout figure), or M(IL-4) (labeled M2 throughout figure) human MDMs, as measured by real-time PCR. Gene expression level is expressed as fold change ± SD relative to M0 condition ( n = 7–8 independent samples, each generated from different donors; each independent sample value corresponds to the average of two technical replicates). One-way analysis of variance (ANOVA) with p values adjusted for multiple comparisons using Sidak’s multiple comparisons test, * p
    Figure Legend Snippet: Increased CD38 expression in human M(LPS + IFN-γ) monocyte-derived macrophages (MDMs). Expression of human CD38 (A) , FPR2 (B) , GPR18 (C) , EGR2 (D) , and c-MYC (E) genes in human MDMs in unstimulated (M0), M(LPS + IFN-γ) (labeled M1 throughout figure), or M(IL-4) (labeled M2 throughout figure) human MDMs, as measured by real-time PCR. Gene expression level is expressed as fold change ± SD relative to M0 condition ( n = 7–8 independent samples, each generated from different donors; each independent sample value corresponds to the average of two technical replicates). One-way analysis of variance (ANOVA) with p values adjusted for multiple comparisons using Sidak’s multiple comparisons test, * p

    Techniques Used: Expressing, Derivative Assay, Labeling, Real-time Polymerase Chain Reaction, Generated

    CD38 mRNA and protein expression is increased in human monocytic cell lines differentiated into M(LPS + IFN-γ) macrophages. Expression of CD38 and FPR2 mRNA in THP-1 (A,B) and U937-derived macrophages (C,D) in unstimulated (M0), M(LPS + IFN-γ)-stimulated (labeled M1 throughout figure), or M(IL-4)-stimulated (labeled M2 throughout figure) macrophages was measured by real-time PCR and expressed as mean relative expression ± SD ( n = 3 biological replicates with two technical replicates per sample). Gene expression is expressed as fold change ± SD relative to M0 condition. One-way analysis of variance with p values adjusted for multiple comparisons using Sidak’s multiple comparisons test compare M(LPS + IFN-γ) vs. M0 and M(LPS + IFN-γ) vs. M(IL-4). (E,F) Flow cytometry staining of surface FPR2 on x -axis, CD38 on y -axis in THP-1 (E) and U937 (F) cells. Flow plots correspond to total cells. Data shown are representative of n = 3 biological replicates. (G–J) Quantification of CD38 + cells and FPR2 + cells in THP-1 (G,H) or U937 cells (I,J) are expressed as percent of positive cells ± SD ( n = 3 biological replicates); ISO, isotype control; ** p
    Figure Legend Snippet: CD38 mRNA and protein expression is increased in human monocytic cell lines differentiated into M(LPS + IFN-γ) macrophages. Expression of CD38 and FPR2 mRNA in THP-1 (A,B) and U937-derived macrophages (C,D) in unstimulated (M0), M(LPS + IFN-γ)-stimulated (labeled M1 throughout figure), or M(IL-4)-stimulated (labeled M2 throughout figure) macrophages was measured by real-time PCR and expressed as mean relative expression ± SD ( n = 3 biological replicates with two technical replicates per sample). Gene expression is expressed as fold change ± SD relative to M0 condition. One-way analysis of variance with p values adjusted for multiple comparisons using Sidak’s multiple comparisons test compare M(LPS + IFN-γ) vs. M0 and M(LPS + IFN-γ) vs. M(IL-4). (E,F) Flow cytometry staining of surface FPR2 on x -axis, CD38 on y -axis in THP-1 (E) and U937 (F) cells. Flow plots correspond to total cells. Data shown are representative of n = 3 biological replicates. (G–J) Quantification of CD38 + cells and FPR2 + cells in THP-1 (G,H) or U937 cells (I,J) are expressed as percent of positive cells ± SD ( n = 3 biological replicates); ISO, isotype control; ** p

    Techniques Used: Expressing, Derivative Assay, Labeling, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Staining

    CD38 mean fluorescence intensity (MFI) in non-classical monocytes (NCMs) is elevated in active systemic lupus erythematosus (SLE). (A) Gating strategy: peripheral blood mononuclear cells isolated from healthy donors (HD) or SLE patients (panel 1) were gated for CD45 + cells (panel 2) prior to gating out CD66b − cells (panel 3) and CD3 − CD19 − CD56 − (panel 4) cells. The remaining population was analyzed with CD14 and CD16 after removing cells that are neither CD14 + nor CD16 + (NOT gate) (panel 5) to identify classical (CD14 ++ CD16 − ) (CM), intermediate (CD14 + CD16 + ) (IM) and non-classical (CD14 + CD16 + ) (NCM) monocytes (panel 6). A stricter gate that excluded CD14 − cells was also used for some analyses (box in panel 6 labeled CD14 low CD16 ++ ). (B) Example of how the relative percentage of CM, IM, and NCM among monocytes was calculated and how CM, IM, and NCM populations were analyzed for CD38 expression. ISO, isotype. (C) Percent of CD38 + cells within the CM, IM and NCM subsets in HD, inactive (SLE I = SLEDAI 0), or active (SLE A = SLEDAI > 4) patients. (D) A histogram of CD38 expression in NCM indicating how the CD38 hi subset was defined. A histogram from each group is overlaid, including an SLE I patient with an SLE Disease Activity Index (SLEDAI) of 0 and an SLE A patient with an SLEDAI of 16. (E) CD38 MFI within CD38 + CM, IM, and NCM populations of HD, SLE I , and SLE A patients. (F) Percent of CD38 hi cells within CM, IM, and NCM subsets in HD, SLE I , or SLE A patients. (G) CD38 MFI within CD38 + CD14 low CD16 ++ population in HD, SLE I , or SLE A patients. (H) Percent of CD38 hi cells within CD14 low CD16 ++ population in HD, SLE I , or SLE A patients. (C,E–H) Quantification of CD38 + cells (C) and CD38 hi cells (F,H) is expressed as percent of positive cells ± SD. Quantification of CD38 MFI (E,G) is expressed as MFI of positive cells ± SD. n = 9 donors for HD, n = 11 patients for SLE I , and n = 10 patients for SLE A . Each sample was run in duplicate, and duplicate values were averaged prior to analysis. One-way analysis of variance with p values adjusted for multiple comparisons using Tukey’s multiple comparisons test, * p
    Figure Legend Snippet: CD38 mean fluorescence intensity (MFI) in non-classical monocytes (NCMs) is elevated in active systemic lupus erythematosus (SLE). (A) Gating strategy: peripheral blood mononuclear cells isolated from healthy donors (HD) or SLE patients (panel 1) were gated for CD45 + cells (panel 2) prior to gating out CD66b − cells (panel 3) and CD3 − CD19 − CD56 − (panel 4) cells. The remaining population was analyzed with CD14 and CD16 after removing cells that are neither CD14 + nor CD16 + (NOT gate) (panel 5) to identify classical (CD14 ++ CD16 − ) (CM), intermediate (CD14 + CD16 + ) (IM) and non-classical (CD14 + CD16 + ) (NCM) monocytes (panel 6). A stricter gate that excluded CD14 − cells was also used for some analyses (box in panel 6 labeled CD14 low CD16 ++ ). (B) Example of how the relative percentage of CM, IM, and NCM among monocytes was calculated and how CM, IM, and NCM populations were analyzed for CD38 expression. ISO, isotype. (C) Percent of CD38 + cells within the CM, IM and NCM subsets in HD, inactive (SLE I = SLEDAI 0), or active (SLE A = SLEDAI > 4) patients. (D) A histogram of CD38 expression in NCM indicating how the CD38 hi subset was defined. A histogram from each group is overlaid, including an SLE I patient with an SLE Disease Activity Index (SLEDAI) of 0 and an SLE A patient with an SLEDAI of 16. (E) CD38 MFI within CD38 + CM, IM, and NCM populations of HD, SLE I , and SLE A patients. (F) Percent of CD38 hi cells within CM, IM, and NCM subsets in HD, SLE I , or SLE A patients. (G) CD38 MFI within CD38 + CD14 low CD16 ++ population in HD, SLE I , or SLE A patients. (H) Percent of CD38 hi cells within CD14 low CD16 ++ population in HD, SLE I , or SLE A patients. (C,E–H) Quantification of CD38 + cells (C) and CD38 hi cells (F,H) is expressed as percent of positive cells ± SD. Quantification of CD38 MFI (E,G) is expressed as MFI of positive cells ± SD. n = 9 donors for HD, n = 11 patients for SLE I , and n = 10 patients for SLE A . Each sample was run in duplicate, and duplicate values were averaged prior to analysis. One-way analysis of variance with p values adjusted for multiple comparisons using Tukey’s multiple comparisons test, * p

    Techniques Used: Fluorescence, Isolation, Labeling, Expressing, Activity Assay

    17) Product Images from "Altered Memory Circulating T Follicular Helper-B Cell Interaction in Early Acute HIV Infection"

    Article Title: Altered Memory Circulating T Follicular Helper-B Cell Interaction in Early Acute HIV Infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005777

    Elevated plasma viral load is also associated with a T cell-independent hyperactivated B cell response. Plasma at day 0 and days 5, 14, 56, 84, 168, 252, 336, 504 and 672 of ART, were analyzed by ELISA for (A) Total IgG and (B) HIV-specific IgG. PBMCs from week 0 Stage 1 and 2 (n = 8) and Stage 3 (n = 7) patients were analyzed by flow cytometry for terminally differentiated plasmablasts identified as CD19 + CD10 - CD20 - CD21 - CD38 hi CD27 hi cells. (C) Plasmablasts from stage 1/2 and stage 3 were compared to each other and (D) together with plasma viral load. For graphs (A) and (B) bars represent mean SEM and statistics were carried out using multiple T tests comparing stage 1/2 to stage 3 at the different time points. Symbols on the graphs represent Stage 1/2 individuals (black circles) and Stage 3 individuals (black squares). For (C) bars represent mean ±SD, symbols on the graphs represent stage 1/2 individuals (black circles) and stage 3 individuals (black squares) and statistics carried out using the Mann-Whitney non-parametric test. For (D) symbols represent stage 1/2 individuals (black circles) and stage 3 individuals (black squares) and correlation data was analyzed using Spearman r test. * P
    Figure Legend Snippet: Elevated plasma viral load is also associated with a T cell-independent hyperactivated B cell response. Plasma at day 0 and days 5, 14, 56, 84, 168, 252, 336, 504 and 672 of ART, were analyzed by ELISA for (A) Total IgG and (B) HIV-specific IgG. PBMCs from week 0 Stage 1 and 2 (n = 8) and Stage 3 (n = 7) patients were analyzed by flow cytometry for terminally differentiated plasmablasts identified as CD19 + CD10 - CD20 - CD21 - CD38 hi CD27 hi cells. (C) Plasmablasts from stage 1/2 and stage 3 were compared to each other and (D) together with plasma viral load. For graphs (A) and (B) bars represent mean SEM and statistics were carried out using multiple T tests comparing stage 1/2 to stage 3 at the different time points. Symbols on the graphs represent Stage 1/2 individuals (black circles) and Stage 3 individuals (black squares). For (C) bars represent mean ±SD, symbols on the graphs represent stage 1/2 individuals (black circles) and stage 3 individuals (black squares) and statistics carried out using the Mann-Whitney non-parametric test. For (D) symbols represent stage 1/2 individuals (black circles) and stage 3 individuals (black squares) and correlation data was analyzed using Spearman r test. * P

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

    18) Product Images from "Deficiency in TNFRSF13B (TACI) expands T-follicular helper and germinal center B cells via increased ICOS-ligand expression but impairs plasma cell survival"

    Article Title: Deficiency in TNFRSF13B (TACI) expands T-follicular helper and germinal center B cells via increased ICOS-ligand expression but impairs plasma cell survival

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

    doi: 10.1073/pnas.1200386109

    Up-regulation of B7H2 expression on Taci −/− B cells. ( A and B ) Histogram depicting B7H2 expression level on GC B cells (CD19 + CD38 − Fas + ) and non-GC B cells (CD19 + CD38 + Fas − ) from spleens of day 10 NP 38 -CCG–immunized
    Figure Legend Snippet: Up-regulation of B7H2 expression on Taci −/− B cells. ( A and B ) Histogram depicting B7H2 expression level on GC B cells (CD19 + CD38 − Fas + ) and non-GC B cells (CD19 + CD38 + Fas − ) from spleens of day 10 NP 38 -CCG–immunized

    Techniques Used: Expressing

    19) Product Images from "Adaptor protein DOK3 promotes plasma cell differentiation by regulating the expression of programmed cell death 1 ligands"

    Article Title: Adaptor protein DOK3 promotes plasma cell differentiation by regulating the expression of programmed cell death 1 ligands

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

    doi: 10.1073/pnas.1400539111

    Expansion of T fh and GC B cells in Dok3 −/− mice. Flow cytometry analysis ( A ) and quantification of the percentage and numbers ( B ) of T fh (CD4 + TCRβ + CXCR5 + PD-1 + ) and GC B (CD19 + CD38 − Fas + ) cells in the Peyer’s patches of unimmunized WT and Dok3 −/− mice. ( C ) Representative flow cytometry analysis of T fh and GC B cells in spleens of WT and Dok3 −/− mice at day 10 after immunization. ( D ) Quantification of T fh and GC B cells in immunized WT and Dok3 −/− mice as shown in C . Numbers indicate percentage of CD4 + cells for T fh cells and CD19 + cells for GC B cells. Each symbol represents one mouse analyzed. * P
    Figure Legend Snippet: Expansion of T fh and GC B cells in Dok3 −/− mice. Flow cytometry analysis ( A ) and quantification of the percentage and numbers ( B ) of T fh (CD4 + TCRβ + CXCR5 + PD-1 + ) and GC B (CD19 + CD38 − Fas + ) cells in the Peyer’s patches of unimmunized WT and Dok3 −/− mice. ( C ) Representative flow cytometry analysis of T fh and GC B cells in spleens of WT and Dok3 −/− mice at day 10 after immunization. ( D ) Quantification of T fh and GC B cells in immunized WT and Dok3 −/− mice as shown in C . Numbers indicate percentage of CD4 + cells for T fh cells and CD19 + cells for GC B cells. Each symbol represents one mouse analyzed. * P

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

    20) Product Images from "Human Cytomegalovirus Induces Cellular and Humoral Virus-specific Immune Responses in Humanized BLT Mice"

    Article Title: Human Cytomegalovirus Induces Cellular and Humoral Virus-specific Immune Responses in Humanized BLT Mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01051-5

    Generation of humanized BLT-NSG (huBLT) mice and human cell reconstitution. ( a ) Bone marrow-Liver-Thymus (huBLT) mice were generated by transplantation of human fetal liver and thymus under the kidney capsule of an adult NSG mouse. Post-surgical transplant, mice were sublethally irradiated (200 cGy) and intravenously injected with human CD34+ hematopoietic progenitor cells (HPCs) isolated from autologous fetal liver tissue. Post T-cell reconstitution (12–16 weeks following engraftment), huBLT mice were infected with HCMV by intraperitoneal (IP) injection of HCMV-infected fibroblasts or Mock infected by IP injection of uninfected fibroblasts. Beginning at 6 weeks post-infection huBLT mice are screened for HCMV-specific immune responses. Latently-infected huBLT mice are treated with G-CSF to induce viral reactivation at 8 weeks post-infection. ( b ) Human cell reconstitution was monitored by flow cytometry analysis of peripheral blood for the percentage of human CD45+ leukocytes (out of total human plus murine CD45+ leukocytes) beginning at 8 weeks post-humanization. Human CD45+ leukocytes can be further analyzed using human specific antibodies, including assessment of CD3+ T-cells and CD19+ B-cells (panel 1) and CD3+ T-cells further discriminated into CD4 and CD8 subsets (panel 2). In addition, monocyte subsets, as characterized by CD14 and CD16 staining, are present in the periphery (panel 3). C) Progenitor cell reconstitution was analyzed in the bone marrow using antibodies for human CD45 and CD34 (panel 1). CD34+ HPCs were further analyzed using antibodies for CD117 (c-kit) and CD38 (panel 2), and monocyte subsets cells analyzed using antibodies for CD33 (early) and CD14 (maturing) (panel 3). Data shown in ( b and c ) is from a huBLT mouse (cohort 3) at 17 weeks post-humanization gated on viable, muCD45- leukocytes. huBLT mice were divided equally into experimental groups based on overall human leukocyte reconstitution (human CD45+) and human T-cell reconstitution (human CD3+) in the periphery. At 8 weeks post-infection, huBLT mice (cohort 2) were reactivated by treatment with G-CSF and AMD3100. Seven days post-reactivation, all mice were euthanized and lymphoid organs collected. Genomic DNA was isolated using DNAzol and viral load determined by qPCR using primers and probe specific for HCMV UL144. Each sample was analyzed in triplicate. Data is shown for the average with standard error of the mean of two spleen tissue sections ( d ) or four liver tissue sections ( e ) per mouse (HCMV, n = 7; HCMV + G-CSF, n = 6), normalized to 1 ug of input DNA. Statistical analysis performed by one-way ANOVA.
    Figure Legend Snippet: Generation of humanized BLT-NSG (huBLT) mice and human cell reconstitution. ( a ) Bone marrow-Liver-Thymus (huBLT) mice were generated by transplantation of human fetal liver and thymus under the kidney capsule of an adult NSG mouse. Post-surgical transplant, mice were sublethally irradiated (200 cGy) and intravenously injected with human CD34+ hematopoietic progenitor cells (HPCs) isolated from autologous fetal liver tissue. Post T-cell reconstitution (12–16 weeks following engraftment), huBLT mice were infected with HCMV by intraperitoneal (IP) injection of HCMV-infected fibroblasts or Mock infected by IP injection of uninfected fibroblasts. Beginning at 6 weeks post-infection huBLT mice are screened for HCMV-specific immune responses. Latently-infected huBLT mice are treated with G-CSF to induce viral reactivation at 8 weeks post-infection. ( b ) Human cell reconstitution was monitored by flow cytometry analysis of peripheral blood for the percentage of human CD45+ leukocytes (out of total human plus murine CD45+ leukocytes) beginning at 8 weeks post-humanization. Human CD45+ leukocytes can be further analyzed using human specific antibodies, including assessment of CD3+ T-cells and CD19+ B-cells (panel 1) and CD3+ T-cells further discriminated into CD4 and CD8 subsets (panel 2). In addition, monocyte subsets, as characterized by CD14 and CD16 staining, are present in the periphery (panel 3). C) Progenitor cell reconstitution was analyzed in the bone marrow using antibodies for human CD45 and CD34 (panel 1). CD34+ HPCs were further analyzed using antibodies for CD117 (c-kit) and CD38 (panel 2), and monocyte subsets cells analyzed using antibodies for CD33 (early) and CD14 (maturing) (panel 3). Data shown in ( b and c ) is from a huBLT mouse (cohort 3) at 17 weeks post-humanization gated on viable, muCD45- leukocytes. huBLT mice were divided equally into experimental groups based on overall human leukocyte reconstitution (human CD45+) and human T-cell reconstitution (human CD3+) in the periphery. At 8 weeks post-infection, huBLT mice (cohort 2) were reactivated by treatment with G-CSF and AMD3100. Seven days post-reactivation, all mice were euthanized and lymphoid organs collected. Genomic DNA was isolated using DNAzol and viral load determined by qPCR using primers and probe specific for HCMV UL144. Each sample was analyzed in triplicate. Data is shown for the average with standard error of the mean of two spleen tissue sections ( d ) or four liver tissue sections ( e ) per mouse (HCMV, n = 7; HCMV + G-CSF, n = 6), normalized to 1 ug of input DNA. Statistical analysis performed by one-way ANOVA.

    Techniques Used: Mouse Assay, Generated, Transplantation Assay, Irradiation, Injection, Isolation, Infection, Flow Cytometry, Cytometry, Staining, Real-time Polymerase Chain Reaction

    huBLT mice develop mature human B-cells and functional HCMV-specific antibody responses. huBLT mice were generated as described in Fig. 1 . Total mononuclear cells from the bone marrow ( a ) and liver ( b ) from an uninfected huBLT mouse at 17 weeks post-humanization were analyzed by flow cytometry for human B-cell subsets. Samples were gated on total viable, murine CD45-, human CD45+ lymphocytes. Both organs were analyzed for B-cell maturation looking at CD10+ CD19- committed lymphoid progenitors (CLP, which are also CD38+ CD20−) and CD10+ CD19+ pre-B-cells and CD10-CD19+ naïve B-cells (panel 1). The CD19+ B-cell population was further analyzed for CD27-CD20- naïve B-cells, CD20 + CD27− immature/naïve mature B-cells, CD27 + CD20+ memory B-cells and CD27 + CD20− plasma B-cells (panel 2). huBLT mice were generated and infected with HCMV as described in Fig. 1 and euthanized at 8 weeks post-infection. Plasma samples were analyzed by ELISA for HCMV antibodies using a pan-IgG/IgM/IgA secondary antibody. Positive samples were re-analyzed for antibody maturation using secondary antibodies specific for IgM ( c ) or IgG ( d ).
    Figure Legend Snippet: huBLT mice develop mature human B-cells and functional HCMV-specific antibody responses. huBLT mice were generated as described in Fig. 1 . Total mononuclear cells from the bone marrow ( a ) and liver ( b ) from an uninfected huBLT mouse at 17 weeks post-humanization were analyzed by flow cytometry for human B-cell subsets. Samples were gated on total viable, murine CD45-, human CD45+ lymphocytes. Both organs were analyzed for B-cell maturation looking at CD10+ CD19- committed lymphoid progenitors (CLP, which are also CD38+ CD20−) and CD10+ CD19+ pre-B-cells and CD10-CD19+ naïve B-cells (panel 1). The CD19+ B-cell population was further analyzed for CD27-CD20- naïve B-cells, CD20 + CD27− immature/naïve mature B-cells, CD27 + CD20+ memory B-cells and CD27 + CD20− plasma B-cells (panel 2). huBLT mice were generated and infected with HCMV as described in Fig. 1 and euthanized at 8 weeks post-infection. Plasma samples were analyzed by ELISA for HCMV antibodies using a pan-IgG/IgM/IgA secondary antibody. Positive samples were re-analyzed for antibody maturation using secondary antibodies specific for IgM ( c ) or IgG ( d ).

    Techniques Used: Mouse Assay, Functional Assay, Generated, Flow Cytometry, Cytometry, Infection, Enzyme-linked Immunosorbent Assay

    21) Product Images from "Alcohol abstinence ameliorates the dysregulated immune profiles in patients with alcoholic hepatitis: a prospective observational study"

    Article Title: Alcohol abstinence ameliorates the dysregulated immune profiles in patients with alcoholic hepatitis: a prospective observational study

    Journal: Hepatology (Baltimore, Md.)

    doi: 10.1002/hep.29242

    Kaplan-Meier survival curves and comparison of immune markers in steroid-treated and untreated AH patients. (A) Kaplan-Meier curves showing 3- and 6-month survival according to baseline CD38 expression levels on monocytes. The mean fluorescent intensity (MFI) of 2,999 was used as the cut-off to define patients with low (n=17) or high (n=18) CD38 expression. (B) Scatter plots of plasma IL-6, IFNα-2, IL-8, MDC levels, CD38-expressing monocytes, and IFN-γ-expressing CD4 T cells in steroid ± pentoxifyllinetreated (Treated) and non-treated (NT) patients. Mann Whitney test for comparisons at days 0 (D0), 180 (D180), and 360 (D360). Horizontal lines represent the median; *p
    Figure Legend Snippet: Kaplan-Meier survival curves and comparison of immune markers in steroid-treated and untreated AH patients. (A) Kaplan-Meier curves showing 3- and 6-month survival according to baseline CD38 expression levels on monocytes. The mean fluorescent intensity (MFI) of 2,999 was used as the cut-off to define patients with low (n=17) or high (n=18) CD38 expression. (B) Scatter plots of plasma IL-6, IFNα-2, IL-8, MDC levels, CD38-expressing monocytes, and IFN-γ-expressing CD4 T cells in steroid ± pentoxifyllinetreated (Treated) and non-treated (NT) patients. Mann Whitney test for comparisons at days 0 (D0), 180 (D180), and 360 (D360). Horizontal lines represent the median; *p

    Techniques Used: Expressing, MANN-WHITNEY

    22) Product Images from "Isolation and Characterisation of Antigen-Specific Plasmablasts Using a Novel Flow Cytometry-Based Immunoglobulin Capture Assay (ICA)"

    Article Title: Isolation and Characterisation of Antigen-Specific Plasmablasts Using a Novel Flow Cytometry-Based Immunoglobulin Capture Assay (ICA)

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

    doi: 10.4049/jimmunol.1701253

    Gating Strategy for Sorting of Antigen Specific IgG+ Plasmablasts. (A) PBMCs were first gated by forward (FSC) and side scatter (SSC), extending the normal lymphocyte gate beyond the standard limits into the monocytes, to account for the increased size and granularity of plasmablasts. These cells were then gated to exclude doublets by comparing the forward scatter area and height measurements. Single cells were gated on live cells shown by only level staining by Zombie NIR Fixable Viability Dye, followed by gating on CD3 - CD14 - CD19 + cells to exclude T cells and monocytes and to gate on CD19 + B cells. Within the B cell population, plasmablasts were identified as CD27 hi CD38 hi , followed by CD20 - . Plasmablasts expressing IgM, IgD, or IgA were gated out since our cloning strategy only incorporated IgG primers. (B) The final IgA - population as shown in (A) was then interrogated for the level of anti-GFP binding, representing the antigen reactivity. Gates were set using either an unvaccinated sample or a fluorescence minus one (FMO) control. The donors shown here were gated using an unvaccinated control donor (left), and showed a range of CN54gp140-specific responses reflecting the time after vaccination these samples were taken. (C) This approach was also tested using 2 other recombinant GFP-tagged antigens; tetanus toxoid C-Fragment (TTX) and hepatitis B surface antigen (HBsAg). All control donor cells were acquired at baseline, while antigen reactive cells were acquired 5-7 days post-booster vaccination.
    Figure Legend Snippet: Gating Strategy for Sorting of Antigen Specific IgG+ Plasmablasts. (A) PBMCs were first gated by forward (FSC) and side scatter (SSC), extending the normal lymphocyte gate beyond the standard limits into the monocytes, to account for the increased size and granularity of plasmablasts. These cells were then gated to exclude doublets by comparing the forward scatter area and height measurements. Single cells were gated on live cells shown by only level staining by Zombie NIR Fixable Viability Dye, followed by gating on CD3 - CD14 - CD19 + cells to exclude T cells and monocytes and to gate on CD19 + B cells. Within the B cell population, plasmablasts were identified as CD27 hi CD38 hi , followed by CD20 - . Plasmablasts expressing IgM, IgD, or IgA were gated out since our cloning strategy only incorporated IgG primers. (B) The final IgA - population as shown in (A) was then interrogated for the level of anti-GFP binding, representing the antigen reactivity. Gates were set using either an unvaccinated sample or a fluorescence minus one (FMO) control. The donors shown here were gated using an unvaccinated control donor (left), and showed a range of CN54gp140-specific responses reflecting the time after vaccination these samples were taken. (C) This approach was also tested using 2 other recombinant GFP-tagged antigens; tetanus toxoid C-Fragment (TTX) and hepatitis B surface antigen (HBsAg). All control donor cells were acquired at baseline, while antigen reactive cells were acquired 5-7 days post-booster vaccination.

    Techniques Used: Staining, Expressing, Clone Assay, Binding Assay, Fluorescence, Recombinant

    23) Product Images from "RNA-binding protein Ptbp1 is essential for BCR-mediated antibody production"

    Article Title: RNA-binding protein Ptbp1 is essential for BCR-mediated antibody production

    Journal: International Immunology

    doi: 10.1093/intimm/dxy077

    Impaired TD responses in P1BKO mice. (A) Histogram depicts the frequency of NP-PE + B220 + Ki67 + proliferative B cells in mice 1 week after immunization with NP-CGG. Left panel, control mice (ctrl); right panel, P1BKO mice. (B) Percentage (left) and number (right) of NP-PE + Ki67 + cells. (C) Representative FACS profiles of NP-specific B220 low CD138 + plasmablasts from mice 1 week after immunization with NP-CGG. (D) Percentages (left) and number (right) of plasmablasts. (E) Typical FACS profiles of NP-specific GC B cells (IgG 1 + /CD38 low ; upper left) and memory B cells (IgG 1 + /CD38 high ; upper right) from mice 2 weeks after immunization with NP-CGG. (F) Percentages (left) and number (right) of GC B cells. (G) Percentages (left) and number (right) of memory B cells. (H) IgG 1 mean fluorescence intensity (MFI) of memory B cells. The mean values of five pairs are shown. Error bars indicate the SD for each sample. * P
    Figure Legend Snippet: Impaired TD responses in P1BKO mice. (A) Histogram depicts the frequency of NP-PE + B220 + Ki67 + proliferative B cells in mice 1 week after immunization with NP-CGG. Left panel, control mice (ctrl); right panel, P1BKO mice. (B) Percentage (left) and number (right) of NP-PE + Ki67 + cells. (C) Representative FACS profiles of NP-specific B220 low CD138 + plasmablasts from mice 1 week after immunization with NP-CGG. (D) Percentages (left) and number (right) of plasmablasts. (E) Typical FACS profiles of NP-specific GC B cells (IgG 1 + /CD38 low ; upper left) and memory B cells (IgG 1 + /CD38 high ; upper right) from mice 2 weeks after immunization with NP-CGG. (F) Percentages (left) and number (right) of GC B cells. (G) Percentages (left) and number (right) of memory B cells. (H) IgG 1 mean fluorescence intensity (MFI) of memory B cells. The mean values of five pairs are shown. Error bars indicate the SD for each sample. * P

    Techniques Used: Mouse Assay, FACS, Fluorescence

    24) Product Images from "Assessing the role of the T-box transcription factor Eomes in B cell differentiation during either Th1 or Th2 cell-biased responses"

    Article Title: Assessing the role of the T-box transcription factor Eomes in B cell differentiation during either Th1 or Th2 cell-biased responses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0208343

    Memory B cell formation in either Th1 or Th2 cell-biased responses proceeds in the absence of Eomes in B cells. Eomes f/f Cd23 cre/+ and littermate controls were immunized with NP-KLH precipitated in alum (A-C) or infected with influenza (D-E) to assess B cell memory formation. (A) Representative plots of antigen-specific memory B cells (NP + IgG1 + CD38 + B cells) in the spleen; representative of 5 mice at d14 and 5 mice at d28 post-immunization. (B) Summary plot of antigen-specific memory B cell frequency. (C) NP-binding IgG1-secreting B cells in the bone marrow was analyzed via ELISpot at d28 post-immunization; data is from 2 experiments, n = 5 per genotype. Error bars indicate mean ± SEM. No significance was detected using the Mann-Whitney non-parametric test. (D-E) HA + CD38 + IgG2c + B cells in the lung (D) or spleen (E) were assessed in influenza-infected mice 28 days post-infection. Data is from 2 experiments, n = 6–8 per genotype for splenic samples and n = 4–5 per genotype for lung samples. Error bars indicate mean ± SEM. No significance was detected using the Mann-Whitney non-parametric test.
    Figure Legend Snippet: Memory B cell formation in either Th1 or Th2 cell-biased responses proceeds in the absence of Eomes in B cells. Eomes f/f Cd23 cre/+ and littermate controls were immunized with NP-KLH precipitated in alum (A-C) or infected with influenza (D-E) to assess B cell memory formation. (A) Representative plots of antigen-specific memory B cells (NP + IgG1 + CD38 + B cells) in the spleen; representative of 5 mice at d14 and 5 mice at d28 post-immunization. (B) Summary plot of antigen-specific memory B cell frequency. (C) NP-binding IgG1-secreting B cells in the bone marrow was analyzed via ELISpot at d28 post-immunization; data is from 2 experiments, n = 5 per genotype. Error bars indicate mean ± SEM. No significance was detected using the Mann-Whitney non-parametric test. (D-E) HA + CD38 + IgG2c + B cells in the lung (D) or spleen (E) were assessed in influenza-infected mice 28 days post-infection. Data is from 2 experiments, n = 6–8 per genotype for splenic samples and n = 4–5 per genotype for lung samples. Error bars indicate mean ± SEM. No significance was detected using the Mann-Whitney non-parametric test.

    Techniques Used: Infection, Mouse Assay, Binding Assay, Enzyme-linked Immunospot, MANN-WHITNEY

    Eomes is not required for germinal center B cell formation or IgG2c isotype switching during influenza infection. Eomes f/f Cd23 cre/+ and littermate controls were infected with HKx31 influenza virus and mediastinal lymph node-derived and splenic B cells were analyzed 8 days post-infection. (A-B) Mature activated B cells (B220 + IgD lo ) were stained to identify germinal center B cells (CD95 hi CD38 lo ). (C-D) The frequency of germinal center B cells that had switched to IgG2c was assessed. (E-F) Frequency of the plasmablast population in either mediastinal lymph nodes (E) or spleen (F). (G-H) Total numbers of germinal center B cells (G) and plasmablasts (H) in the mediastinal lymph node. Data are pooled from 3 experiments, n = 8–10 per genotype. Error bars indicate mean ± SEM. No significant difference was detected using the Mann-Whitney non-parametric test.
    Figure Legend Snippet: Eomes is not required for germinal center B cell formation or IgG2c isotype switching during influenza infection. Eomes f/f Cd23 cre/+ and littermate controls were infected with HKx31 influenza virus and mediastinal lymph node-derived and splenic B cells were analyzed 8 days post-infection. (A-B) Mature activated B cells (B220 + IgD lo ) were stained to identify germinal center B cells (CD95 hi CD38 lo ). (C-D) The frequency of germinal center B cells that had switched to IgG2c was assessed. (E-F) Frequency of the plasmablast population in either mediastinal lymph nodes (E) or spleen (F). (G-H) Total numbers of germinal center B cells (G) and plasmablasts (H) in the mediastinal lymph node. Data are pooled from 3 experiments, n = 8–10 per genotype. Error bars indicate mean ± SEM. No significant difference was detected using the Mann-Whitney non-parametric test.

    Techniques Used: Infection, Derivative Assay, Staining, MANN-WHITNEY

    25) Product Images from "Shp1 signalling is required to establish the long-lived bone marrow plasma cell pool"

    Article Title: Shp1 signalling is required to establish the long-lived bone marrow plasma cell pool

    Journal: Nature Communications

    doi: 10.1038/ncomms5273

    Analyses of GC and memory B-cell formation in Ptpn6 f/f Aicda Cre/+ mice. Ptpn6 +/+ Aicda Cre/+ and Ptpn6 f/f Aicda Cre/+ mice were analysed 10 days onwards after challenge with NP 38 -CGG. ( a ) Flow cytometry analysis of CD19 + cells in the spleens of immunized mice stained for the presence of Fas + CD38 − GC B cells. Numbers indicate per cent of CD19 + cells. ( b ) Graphical depiction of the frequencies of GC B cells in the spleens of Ptpn6 +/+ Aicda Cre/+ and Ptpn6 f/f Aicda Cre/+ mice. Each data point represents one mouse analysed and five mice per group were examined; ns=non-significant. ( c ) Histology staining of splenic GC with anti-IgD (green) and anti-GL7 (red) antibodies. White bar, 200 μm. ( d ) B220 + Dump − cells in the spleens of immunized mice were analysed for the presence of antigen-specific NIP-binding IgG1 B cells. Numbers indicate per cent of IgG1 B cells in B220 + Dump − gate that bind antigen. ( e ) Graphical representation of the frequencies of antigen-specific IgG1 cells in immunized spleens. ( f ) Graphical representation of the frequency of antigen-specific IgG1 cells at day 10, 14 and 28 post immunizations. Cells were gated as in ( d ). Group of five mice were used, and data are expressed as mean±s.e.m. ( g ) Flow cytometry analysis of NIP-binding IgG1 cells at day 14 of immunization to further delineate CD38 − GC and CD38 + memory B cells. Numbers indicate actual number of cells in the gated areas out of 10 4 B220 + Dump − spleen cells analysed. ( h ) Quantification of CD38 + IgG1 + NP-specific memory B cells (expressed as number of memory B cells per 10 4 B220 + Dump − cells) in Ptpn6 +/+ Aicda Cre/+ and Ptpn6 f/f Aicda Cre/+ mice at 0, 14 and 28 days post immunizations. Group of five mice were used, and data are expressed as mean±s.e.m.
    Figure Legend Snippet: Analyses of GC and memory B-cell formation in Ptpn6 f/f Aicda Cre/+ mice. Ptpn6 +/+ Aicda Cre/+ and Ptpn6 f/f Aicda Cre/+ mice were analysed 10 days onwards after challenge with NP 38 -CGG. ( a ) Flow cytometry analysis of CD19 + cells in the spleens of immunized mice stained for the presence of Fas + CD38 − GC B cells. Numbers indicate per cent of CD19 + cells. ( b ) Graphical depiction of the frequencies of GC B cells in the spleens of Ptpn6 +/+ Aicda Cre/+ and Ptpn6 f/f Aicda Cre/+ mice. Each data point represents one mouse analysed and five mice per group were examined; ns=non-significant. ( c ) Histology staining of splenic GC with anti-IgD (green) and anti-GL7 (red) antibodies. White bar, 200 μm. ( d ) B220 + Dump − cells in the spleens of immunized mice were analysed for the presence of antigen-specific NIP-binding IgG1 B cells. Numbers indicate per cent of IgG1 B cells in B220 + Dump − gate that bind antigen. ( e ) Graphical representation of the frequencies of antigen-specific IgG1 cells in immunized spleens. ( f ) Graphical representation of the frequency of antigen-specific IgG1 cells at day 10, 14 and 28 post immunizations. Cells were gated as in ( d ). Group of five mice were used, and data are expressed as mean±s.e.m. ( g ) Flow cytometry analysis of NIP-binding IgG1 cells at day 14 of immunization to further delineate CD38 − GC and CD38 + memory B cells. Numbers indicate actual number of cells in the gated areas out of 10 4 B220 + Dump − spleen cells analysed. ( h ) Quantification of CD38 + IgG1 + NP-specific memory B cells (expressed as number of memory B cells per 10 4 B220 + Dump − cells) in Ptpn6 +/+ Aicda Cre/+ and Ptpn6 f/f Aicda Cre/+ mice at 0, 14 and 28 days post immunizations. Group of five mice were used, and data are expressed as mean±s.e.m.

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Staining, Binding Assay

    26) Product Images from "The Incubation Period of Primary Epstein-Barr Virus Infection: Viral Dynamics and Immunologic Events"

    Article Title: The Incubation Period of Primary Epstein-Barr Virus Infection: Viral Dynamics and Immunologic Events

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005286

    CD8 T cell activation occurred during the incubation period, although not an EBV specific response. (A), Time to first response for three distinct immune parameters is shown: CD38 upregulation on total CD8 + T cells (filled squares), an increased CD8 to CD4 T cell ratio (open diamonds), or the presence of EBV tetramer binding CD8 + T cells above background (0.4%) (filled circles). (B) Frequency of CD8 + T cells expressing CD38 over time. (C) Ratio of CD8 + to CD4 + T cells over time. Statistics were performed using a one-way ANOVA with multiple test comparison. Pink symbols indicate a significant difference (p
    Figure Legend Snippet: CD8 T cell activation occurred during the incubation period, although not an EBV specific response. (A), Time to first response for three distinct immune parameters is shown: CD38 upregulation on total CD8 + T cells (filled squares), an increased CD8 to CD4 T cell ratio (open diamonds), or the presence of EBV tetramer binding CD8 + T cells above background (0.4%) (filled circles). (B) Frequency of CD8 + T cells expressing CD38 over time. (C) Ratio of CD8 + to CD4 + T cells over time. Statistics were performed using a one-way ANOVA with multiple test comparison. Pink symbols indicate a significant difference (p

    Techniques Used: Activation Assay, Incubation, Binding Assay, Expressing

    27) Product Images from "Lactobacillus johnsonii N6.2 Modulates the Host Immune Responses: A Double-Blind, Randomized Trial in Healthy Adults"

    Article Title: Lactobacillus johnsonii N6.2 Modulates the Host Immune Responses: A Double-Blind, Randomized Trial in Healthy Adults

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00655

    T cell subset. CD4 + (A) or CD8 + (B) T cells populations subsets [Naïve, Tem, activated (CD38 + HLA-DR + )] were quantified after 8 or 12 weeks of treatment in the placebo and L. johnsonii N6.2 (Ljo) groups. Naïve (CD197 + CD45RA + ), Tem (CD197 − CD45RA − ), Tcm (CD197 + CD45RA − ), and Temra (CD197 − CD45RA + ) by labeling with specific antibodies (C) . The concentration of cells shown has been normalized to the concentration found at time 0 for each subject.
    Figure Legend Snippet: T cell subset. CD4 + (A) or CD8 + (B) T cells populations subsets [Naïve, Tem, activated (CD38 + HLA-DR + )] were quantified after 8 or 12 weeks of treatment in the placebo and L. johnsonii N6.2 (Ljo) groups. Naïve (CD197 + CD45RA + ), Tem (CD197 − CD45RA − ), Tcm (CD197 + CD45RA − ), and Temra (CD197 − CD45RA + ) by labeling with specific antibodies (C) . The concentration of cells shown has been normalized to the concentration found at time 0 for each subject.

    Techniques Used: Transmission Electron Microscopy, Labeling, Concentration Assay

    T effector cells subset (CD3 + CD4 + CD45RO + ). (A) Th1 (CD183 + CD196 − ), Th2 (CD183 − CD196 − ), Th17 (CD183 − CD196 + ), and Th1/Th17 (CD183 + CD196 + ) were labeled with specific antibodies and quantified in the placebo (white bars) and L. johnsonii N6.2 (Ljo, blue bars) group at 8 and 12 weeks of treatment. (B) HLA-DR + and (C) HLA-DR + CD38 + are shown for the Th1 and Th1/Th17 effector T cells. The concentration of cells shown has been normalized to the concentration found at time 0 for each subject.
    Figure Legend Snippet: T effector cells subset (CD3 + CD4 + CD45RO + ). (A) Th1 (CD183 + CD196 − ), Th2 (CD183 − CD196 − ), Th17 (CD183 − CD196 + ), and Th1/Th17 (CD183 + CD196 + ) were labeled with specific antibodies and quantified in the placebo (white bars) and L. johnsonii N6.2 (Ljo, blue bars) group at 8 and 12 weeks of treatment. (B) HLA-DR + and (C) HLA-DR + CD38 + are shown for the Th1 and Th1/Th17 effector T cells. The concentration of cells shown has been normalized to the concentration found at time 0 for each subject.

    Techniques Used: Labeling, Concentration Assay

    Related Articles

    Flow Cytometry:

    Article Title: African-centric TP53 variant increases iron accumulation and bacterial pathogenesis but improves response to malaria toxin
    Article Snippet: .. Antibodies for flow cytometry Anti CD4 Rat Monoclonal PE/Dazzle 594 (BioLegend Cat # 100566), Anti CD8a Rat Monoclonal APC-H7 (BD Biosciences Cat # 560182), Anti CD25 Rat Monoclonal APC (BioLegend Cat #101909), Anti CD69 Armenian Hamster Monoclonal BV711(BioLegend Cat # 104537), Anti Foxp3 Rat Monoclonal PE (BD BiosciencesCat # 560414), Anti CD11b Rat Monoclonal APC Cy7 (BD Biosciences Cat # 557657), Anti F4/80 Rat Monoclonal APC (BioLegend Cat # 123116), Anti Gr-1 Rat Monoclonal BV711 (BioLegend Cat # 108443), Anti CD38 Rat Monoclonal PE/Cy7 (BioLegend Cat # 102717), Anti EGR2 Rat Monoclonal (PE Life Tech Cat # 12-6691-80). .. Proteomics Protein samples were concentrated (up to eightfold) by lyophilization and 13 μg from each sample was separated by SDS–PAGE for a distance of 1.5 cm.

    Cytometry:

    Article Title: African-centric TP53 variant increases iron accumulation and bacterial pathogenesis but improves response to malaria toxin
    Article Snippet: .. Antibodies for flow cytometry Anti CD4 Rat Monoclonal PE/Dazzle 594 (BioLegend Cat # 100566), Anti CD8a Rat Monoclonal APC-H7 (BD Biosciences Cat # 560182), Anti CD25 Rat Monoclonal APC (BioLegend Cat #101909), Anti CD69 Armenian Hamster Monoclonal BV711(BioLegend Cat # 104537), Anti Foxp3 Rat Monoclonal PE (BD BiosciencesCat # 560414), Anti CD11b Rat Monoclonal APC Cy7 (BD Biosciences Cat # 557657), Anti F4/80 Rat Monoclonal APC (BioLegend Cat # 123116), Anti Gr-1 Rat Monoclonal BV711 (BioLegend Cat # 108443), Anti CD38 Rat Monoclonal PE/Cy7 (BioLegend Cat # 102717), Anti EGR2 Rat Monoclonal (PE Life Tech Cat # 12-6691-80). .. Proteomics Protein samples were concentrated (up to eightfold) by lyophilization and 13 μg from each sample was separated by SDS–PAGE for a distance of 1.5 cm.

    Activation Assay:

    Article Title: Robust Reconstitution of Tuberculosis-Specific Polyfunctional CD4+ T-Cell Responses and Rising Systemic Interleukin 6 in Paradoxical Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome
    Article Snippet: .. T-cell activation was determined with anti-CD38-PE-Cy7 (Biolegend), anti-HLA-DR-BV785 or FITC (Biolegend, BD Biosciences), and anti-PD1-APC-Cy7 (Biolegend). .. Intracellular cytokine staining was performed with anti-IFN-γ-Pacific blue (Biolegend), anti-IL-2-AF647 (Biolegend), and anti-TNF-α-AF700 (eBioscience).

    Expressing:

    Article Title: Signal inhibition by the dual-specific phosphatase 4 impairs T cell-dependent B-cell responses with age
    Article Snippet: .. Splenocytes were analyzed for cell surface marker expression using the following antibodies: PE-CD4, APC-CD62L, APC-CD154 (CD40L), PE-B220, and APC-streptavidin (eBioscience), Alexa Fluor 647-CD278 (ICOS), PerCP/Cy5.5-CD150 (SLAM), and PE/Cy7-CD38 (BioLegend), PerCP-B220, PerCP/Cy5.5-CD44, and PE/Cy7-CXCR5 (BD Pharmingen) as well as biotin-peanut agglutinin (Vector Laboratories) and NP-PE (Biosearch Technologies). .. NP-OVA-specific IgG was quantified by the mouse IgG ELISA quantitation kit (Bethyl Laboratories) using NP-OVA (10 μg/mL) as the capture antigen.

    Marker:

    Article Title: Immunisation of two rodent species with new live-attenuated mutants of Yersinia pestis CO92 induces protective long-term humoral- and cell-mediated immunity against pneumonic plague
    Article Snippet: .. The surface of the B cells was stained with monoclonal anti-mouse CD19-FITC (B-cell surface marker; BioLegend), anti-mouse CD38-PE/Cy7 (memory B-cell marker; BioLegend) and anti-mouse IgG-PE (mature, isotype-switched B-cell marker; Southern Biotech; Birmingham, AL) for 30 min in the dark at 4 °C. .. To measure T cell kinetics, splenic cells were pretreated with ionomycin (750 ng/ml) and phorbol 12- myristate 13-acetate (PMA, 50 ng per sample), and then incubated 2 h later with Brefeldin A (0.7 μg per sample) to accumulate intracellular cytokines.

    Article Title: Signal inhibition by the dual-specific phosphatase 4 impairs T cell-dependent B-cell responses with age
    Article Snippet: .. Splenocytes were analyzed for cell surface marker expression using the following antibodies: PE-CD4, APC-CD62L, APC-CD154 (CD40L), PE-B220, and APC-streptavidin (eBioscience), Alexa Fluor 647-CD278 (ICOS), PerCP/Cy5.5-CD150 (SLAM), and PE/Cy7-CD38 (BioLegend), PerCP-B220, PerCP/Cy5.5-CD44, and PE/Cy7-CXCR5 (BD Pharmingen) as well as biotin-peanut agglutinin (Vector Laboratories) and NP-PE (Biosearch Technologies). .. NP-OVA-specific IgG was quantified by the mouse IgG ELISA quantitation kit (Bethyl Laboratories) using NP-OVA (10 μg/mL) as the capture antigen.

    Staining:

    Article Title: Diminished HIV Infection of Target CD4+ T Cells in a Toll-Like Receptor 4 Stimulated in vitro Model
    Article Snippet: .. The intracellular staining cocktail consisted of anti-CCR5-APC, anti-HLA-DR-PerCP-CY5.5 (all from BD Biosciences, Franklin Lakes, NJ, USA), anti-CD38-PE-CY7 (Biolegend, San Diego, CA, USA) and anti-p24-FITC (Beckman Coulter, Brea, CA, USA). .. PBMCs were collected at two time-points: day 3 (48 h post stimulation and prior to HIV infection) and day 5 (48 h post infection).

    Article Title: Immunisation of two rodent species with new live-attenuated mutants of Yersinia pestis CO92 induces protective long-term humoral- and cell-mediated immunity against pneumonic plague
    Article Snippet: .. The surface of the B cells was stained with monoclonal anti-mouse CD19-FITC (B-cell surface marker; BioLegend), anti-mouse CD38-PE/Cy7 (memory B-cell marker; BioLegend) and anti-mouse IgG-PE (mature, isotype-switched B-cell marker; Southern Biotech; Birmingham, AL) for 30 min in the dark at 4 °C. .. To measure T cell kinetics, splenic cells were pretreated with ionomycin (750 ng/ml) and phorbol 12- myristate 13-acetate (PMA, 50 ng per sample), and then incubated 2 h later with Brefeldin A (0.7 μg per sample) to accumulate intracellular cytokines.

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    BioLegend anti cd38
    <t>CD38</t> expression in peripheral blood
    Anti Cd38, supplied by BioLegend, used in various techniques. Bioz Stars score: 92/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    BioLegend cd38
    BC133 did not cross react with CD34 + <t>CD38</t> – HSCs. (A) Cord blood mononuclear cells were purified using Ficoll-Paque density gradient centrifugation and were immunostained with anti-human CD3, CD19, CD38, CD34, and BC133 antibodies. To exclude T cells and B cells from analysis, cells were gated on CD3 – and CD19 – populations. Different populations of cells (labeled 1 to 6) were assessed for their binding to BC133. (B) HSCs and progenitor cells were isolated from cord blood mononuclear cells using Miltenyi CD34 Microbeads. (C) TDCC by ATC (E:T ratio, 10) in the presence of BC133 against the purified CD34 + cells and MOLM13 AML cells was tested by using chromium release assay.
    Cd38, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    CD38 expression in peripheral blood

    Journal: Clinical and Experimental Immunology

    Article Title: CD38 and E2F transcription factor 2 have uniquely increased expression in rheumatoid arthritis synovial tissues

    doi: 10.1111/cei.12268

    Figure Lengend Snippet: CD38 expression in peripheral blood

    Article Snippet: The following antibodies were used: anti-CD3 labelled with fluorescein isothiocyanate (FITC) (Beckman Coulter, Brea, CA, USA), anti-CD4 labelled with FITC (Beckman Coulter), anti-CD19 labelled with phycoerythrin (PE) (Beckman Coulter), anti-CD22 labelled with FITC (Beckman Coulter), anti-CD25 labelled with PE (Beckman Coulter), anti-CD38 labelled with peridinin chlorophyll cyanin 5·5 (PerCp-Cy5·5) (Biolegend, San Diego, USA) and anti-CD56 labelled with PE (Beckman Coulter).

    Techniques: Expressing

    Tumour necrosis factor (TNF)-α, interleukin (IL)-1α and IL-β levels in the supernatant of siRNA (50 nM)-treated RA synovial fibroblasts RASFs. (a) The CD38 expression was analysed using Western blotting. (b) The E2F transcription

    Journal: Clinical and Experimental Immunology

    Article Title: CD38 and E2F transcription factor 2 have uniquely increased expression in rheumatoid arthritis synovial tissues

    doi: 10.1111/cei.12268

    Figure Lengend Snippet: Tumour necrosis factor (TNF)-α, interleukin (IL)-1α and IL-β levels in the supernatant of siRNA (50 nM)-treated RA synovial fibroblasts RASFs. (a) The CD38 expression was analysed using Western blotting. (b) The E2F transcription

    Article Snippet: The following antibodies were used: anti-CD3 labelled with fluorescein isothiocyanate (FITC) (Beckman Coulter, Brea, CA, USA), anti-CD4 labelled with FITC (Beckman Coulter), anti-CD19 labelled with phycoerythrin (PE) (Beckman Coulter), anti-CD22 labelled with FITC (Beckman Coulter), anti-CD25 labelled with PE (Beckman Coulter), anti-CD38 labelled with peridinin chlorophyll cyanin 5·5 (PerCp-Cy5·5) (Biolegend, San Diego, USA) and anti-CD56 labelled with PE (Beckman Coulter).

    Techniques: Expressing, Western Blot

    Immunohistochemistry of CD38 and E2F transcription factor 2 (E2F2) in synovial membranes from patients with rheumatoid arthritis (RA). CD38 was immunostained in synovial membranes of patients with RA (a–c), osteoarthritis (OA) (d) and

    Journal: Clinical and Experimental Immunology

    Article Title: CD38 and E2F transcription factor 2 have uniquely increased expression in rheumatoid arthritis synovial tissues

    doi: 10.1111/cei.12268

    Figure Lengend Snippet: Immunohistochemistry of CD38 and E2F transcription factor 2 (E2F2) in synovial membranes from patients with rheumatoid arthritis (RA). CD38 was immunostained in synovial membranes of patients with RA (a–c), osteoarthritis (OA) (d) and

    Article Snippet: The following antibodies were used: anti-CD3 labelled with fluorescein isothiocyanate (FITC) (Beckman Coulter, Brea, CA, USA), anti-CD4 labelled with FITC (Beckman Coulter), anti-CD19 labelled with phycoerythrin (PE) (Beckman Coulter), anti-CD22 labelled with FITC (Beckman Coulter), anti-CD25 labelled with PE (Beckman Coulter), anti-CD38 labelled with peridinin chlorophyll cyanin 5·5 (PerCp-Cy5·5) (Biolegend, San Diego, USA) and anti-CD56 labelled with PE (Beckman Coulter).

    Techniques: Immunohistochemistry

    BC133 did not cross react with CD34 + CD38 – HSCs. (A) Cord blood mononuclear cells were purified using Ficoll-Paque density gradient centrifugation and were immunostained with anti-human CD3, CD19, CD38, CD34, and BC133 antibodies. To exclude T cells and B cells from analysis, cells were gated on CD3 – and CD19 – populations. Different populations of cells (labeled 1 to 6) were assessed for their binding to BC133. (B) HSCs and progenitor cells were isolated from cord blood mononuclear cells using Miltenyi CD34 Microbeads. (C) TDCC by ATC (E:T ratio, 10) in the presence of BC133 against the purified CD34 + cells and MOLM13 AML cells was tested by using chromium release assay.

    Journal: Blood Advances

    Article Title: A potent tetravalent T-cell–engaging bispecific antibody against CD33 in acute myeloid leukemia

    doi: 10.1182/bloodadvances.2017014373

    Figure Lengend Snippet: BC133 did not cross react with CD34 + CD38 – HSCs. (A) Cord blood mononuclear cells were purified using Ficoll-Paque density gradient centrifugation and were immunostained with anti-human CD3, CD19, CD38, CD34, and BC133 antibodies. To exclude T cells and B cells from analysis, cells were gated on CD3 – and CD19 – populations. Different populations of cells (labeled 1 to 6) were assessed for their binding to BC133. (B) HSCs and progenitor cells were isolated from cord blood mononuclear cells using Miltenyi CD34 Microbeads. (C) TDCC by ATC (E:T ratio, 10) in the presence of BC133 against the purified CD34 + cells and MOLM13 AML cells was tested by using chromium release assay.

    Article Snippet: Antihuman antibodies against CD34, CD3, CD4, CD8, CD38, CD33, CCR7, CD45RO, and CD127 (BioLegend) were used to stain cells.

    Techniques: Purification, Gradient Centrifugation, Labeling, Binding Assay, Isolation, Release Assay

    Memory B cell formation in either Th1 or Th2 cell-biased responses proceeds in the absence of Eomes in B cells. Eomes f/f Cd23 cre/+ and littermate controls were immunized with NP-KLH precipitated in alum (A-C) or infected with influenza (D-E) to assess B cell memory formation. (A) Representative plots of antigen-specific memory B cells (NP + IgG1 + CD38 + B cells) in the spleen; representative of 5 mice at d14 and 5 mice at d28 post-immunization. (B) Summary plot of antigen-specific memory B cell frequency. (C) NP-binding IgG1-secreting B cells in the bone marrow was analyzed via ELISpot at d28 post-immunization; data is from 2 experiments, n = 5 per genotype. Error bars indicate mean ± SEM. No significance was detected using the Mann-Whitney non-parametric test. (D-E) HA + CD38 + IgG2c + B cells in the lung (D) or spleen (E) were assessed in influenza-infected mice 28 days post-infection. Data is from 2 experiments, n = 6–8 per genotype for splenic samples and n = 4–5 per genotype for lung samples. Error bars indicate mean ± SEM. No significance was detected using the Mann-Whitney non-parametric test.

    Journal: PLoS ONE

    Article Title: Assessing the role of the T-box transcription factor Eomes in B cell differentiation during either Th1 or Th2 cell-biased responses

    doi: 10.1371/journal.pone.0208343

    Figure Lengend Snippet: Memory B cell formation in either Th1 or Th2 cell-biased responses proceeds in the absence of Eomes in B cells. Eomes f/f Cd23 cre/+ and littermate controls were immunized with NP-KLH precipitated in alum (A-C) or infected with influenza (D-E) to assess B cell memory formation. (A) Representative plots of antigen-specific memory B cells (NP + IgG1 + CD38 + B cells) in the spleen; representative of 5 mice at d14 and 5 mice at d28 post-immunization. (B) Summary plot of antigen-specific memory B cell frequency. (C) NP-binding IgG1-secreting B cells in the bone marrow was analyzed via ELISpot at d28 post-immunization; data is from 2 experiments, n = 5 per genotype. Error bars indicate mean ± SEM. No significance was detected using the Mann-Whitney non-parametric test. (D-E) HA + CD38 + IgG2c + B cells in the lung (D) or spleen (E) were assessed in influenza-infected mice 28 days post-infection. Data is from 2 experiments, n = 6–8 per genotype for splenic samples and n = 4–5 per genotype for lung samples. Error bars indicate mean ± SEM. No significance was detected using the Mann-Whitney non-parametric test.

    Article Snippet: The following antibodies were used for flow cytometry: CD95 (JO2), IgG1 (X56), CD138 (281–2), IgG2a (R2-40), IgD (11-26c.2a), CD8 (53–6.7), CD44 (IM & ), FVS fixable viability stains from BD; B220 (RA36B2), IgD (11-26c.2a), CD38 (90) and CD138 (281–2) from Biolegend; CD4 (GK1.5–7) and NIP were conjugated in-house.

    Techniques: Infection, Mouse Assay, Binding Assay, Enzyme-linked Immunospot, MANN-WHITNEY

    Eomes is not required for germinal center B cell formation or IgG2c isotype switching during influenza infection. Eomes f/f Cd23 cre/+ and littermate controls were infected with HKx31 influenza virus and mediastinal lymph node-derived and splenic B cells were analyzed 8 days post-infection. (A-B) Mature activated B cells (B220 + IgD lo ) were stained to identify germinal center B cells (CD95 hi CD38 lo ). (C-D) The frequency of germinal center B cells that had switched to IgG2c was assessed. (E-F) Frequency of the plasmablast population in either mediastinal lymph nodes (E) or spleen (F). (G-H) Total numbers of germinal center B cells (G) and plasmablasts (H) in the mediastinal lymph node. Data are pooled from 3 experiments, n = 8–10 per genotype. Error bars indicate mean ± SEM. No significant difference was detected using the Mann-Whitney non-parametric test.

    Journal: PLoS ONE

    Article Title: Assessing the role of the T-box transcription factor Eomes in B cell differentiation during either Th1 or Th2 cell-biased responses

    doi: 10.1371/journal.pone.0208343

    Figure Lengend Snippet: Eomes is not required for germinal center B cell formation or IgG2c isotype switching during influenza infection. Eomes f/f Cd23 cre/+ and littermate controls were infected with HKx31 influenza virus and mediastinal lymph node-derived and splenic B cells were analyzed 8 days post-infection. (A-B) Mature activated B cells (B220 + IgD lo ) were stained to identify germinal center B cells (CD95 hi CD38 lo ). (C-D) The frequency of germinal center B cells that had switched to IgG2c was assessed. (E-F) Frequency of the plasmablast population in either mediastinal lymph nodes (E) or spleen (F). (G-H) Total numbers of germinal center B cells (G) and plasmablasts (H) in the mediastinal lymph node. Data are pooled from 3 experiments, n = 8–10 per genotype. Error bars indicate mean ± SEM. No significant difference was detected using the Mann-Whitney non-parametric test.

    Article Snippet: The following antibodies were used for flow cytometry: CD95 (JO2), IgG1 (X56), CD138 (281–2), IgG2a (R2-40), IgD (11-26c.2a), CD8 (53–6.7), CD44 (IM & ), FVS fixable viability stains from BD; B220 (RA36B2), IgD (11-26c.2a), CD38 (90) and CD138 (281–2) from Biolegend; CD4 (GK1.5–7) and NIP were conjugated in-house.

    Techniques: Infection, Derivative Assay, Staining, MANN-WHITNEY

    CD43s is overexpressed by myeloid malignancies. (A) AT1413 binding to CD34+ and CD38+ CD45dim AML blasts of patient 101. Bone marrow cells of this patient were isolated using a ficoll gradient and stored at diagnosis, precluding analysis of AT1413 interaction with nonmalignant granulocytes. (B) Representative examples of AT1413 binding to AML blasts obtained from newly diagnosed patients with AML or MDS . (C) AT1413 binding to extramedullary AML of 2 patients (myeloid sarcoma [chloroma] of inguinal node [1] and skin [2]). Paraffin-embedded THP-1 and Jurkat cells were used as a positive and negative control, respectively. Biotin immunoreactivity of antibody shown with streptavidin-HRP and the peroxidase substrate DAB. Scale bars, 20 μm. (D) Bone marrow of a patient with concomitant multiple myeloma and therapy-related AML. (Left) Hematoxylin and eosin staining. Asterisk, malignant double-nucleated plasma cell; arrowheads, AML blasts. Original magnification ×100. (Right) AT1413 staining of CD45dim AML blasts; CD138+ multiple myeloma plasma cells do not interact with AT1413. (E) AT1413 binding to CD45dim blasts of patients with AML, and to a lesser extent to CD45+ granulocytes and monocytes and absence of binding to CD45+ lymphocytes. The fold increase MFI of AT1413 compared with the negative control is indicated in gray (AT1002, filled gray histogram). Bone marrow (BL-079, BL-092, BL-095, BL-096, BL-099) or blood (BL-091, BL-106) of patients with AML was freshly obtained and red blood cells lysed before FACs analysis. RAEB, refractory anemia with excess blasts.

    Journal: Blood Advances

    Article Title: Patient-derived antibody recognizes a unique CD43 epitope expressed on all AML and has antileukemia activity in mice

    doi: 10.1182/bloodadvances.2017008342

    Figure Lengend Snippet: CD43s is overexpressed by myeloid malignancies. (A) AT1413 binding to CD34+ and CD38+ CD45dim AML blasts of patient 101. Bone marrow cells of this patient were isolated using a ficoll gradient and stored at diagnosis, precluding analysis of AT1413 interaction with nonmalignant granulocytes. (B) Representative examples of AT1413 binding to AML blasts obtained from newly diagnosed patients with AML or MDS . (C) AT1413 binding to extramedullary AML of 2 patients (myeloid sarcoma [chloroma] of inguinal node [1] and skin [2]). Paraffin-embedded THP-1 and Jurkat cells were used as a positive and negative control, respectively. Biotin immunoreactivity of antibody shown with streptavidin-HRP and the peroxidase substrate DAB. Scale bars, 20 μm. (D) Bone marrow of a patient with concomitant multiple myeloma and therapy-related AML. (Left) Hematoxylin and eosin staining. Asterisk, malignant double-nucleated plasma cell; arrowheads, AML blasts. Original magnification ×100. (Right) AT1413 staining of CD45dim AML blasts; CD138+ multiple myeloma plasma cells do not interact with AT1413. (E) AT1413 binding to CD45dim blasts of patients with AML, and to a lesser extent to CD45+ granulocytes and monocytes and absence of binding to CD45+ lymphocytes. The fold increase MFI of AT1413 compared with the negative control is indicated in gray (AT1002, filled gray histogram). Bone marrow (BL-079, BL-092, BL-095, BL-096, BL-099) or blood (BL-091, BL-106) of patients with AML was freshly obtained and red blood cells lysed before FACs analysis. RAEB, refractory anemia with excess blasts.

    Article Snippet: The following antibodies were used: IgG H+L AF647 (Life Technologies), IgG Fcy AF647 (Jackson), Dapi (Sigma), the CD43 antibodies 84-3C1 (-PE; Ebioscience), L10 (-FITC; Invitrogen), MEM-59 (unlabeled or -FITC; Abcam), DF-T1 (unlabeled; Thermo Scientific), CD4, CD8, CD14, CD19, CD34, CD38, CD45, and CD66b (Biolegend).

    Techniques: Binding Assay, Isolation, Negative Control, Staining, FACS