anti cd40  (BioLegend)

 
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  • 95
    Name:
    Purified anti mouse CD40
    Description:
    Purified anti mouse CD40 1C10 Isotype Rat IgG2a κ Reactivity Mouse Apps FC IP Size 500 μg
    Catalog Number:
    102802
    Price:
    200
    Category:
    Mouse Immunology
    Source:
    Rat
    Applications:
    FC, IP
    Conjugate:
    PURE
    Size:
    500 μg
    Quantity:
    1
    Preparation:
    The antibody was purified by affinity chromatography
    Buy from Supplier


    Structured Review

    BioLegend anti cd40
    Purified anti mouse CD40
    Purified anti mouse CD40 1C10 Isotype Rat IgG2a κ Reactivity Mouse Apps FC IP Size 500 μg
    https://www.bioz.com/result/anti cd40/product/BioLegend
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti cd40 - by Bioz Stars, 2021-05
    95/100 stars

    Images

    1) Product Images from "Activated group 3 innate lymphoid cells promote T-cell–mediated immune responses"

    Article Title: Activated group 3 innate lymphoid cells promote T-cell–mediated immune responses

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

    doi: 10.1073/pnas.1406908111

    IL-1β induces the expression of MHC class II and costimulatory molecules on peripheral NCR − ILC3s. Expression of CD69, CD40, CD80, CD86, and MHC class II on sort-purified in vitro-generated CD4 + NCR − ILC3s ( A ) and ex vivo-isolated splenic CD4 + NCR − ILC3s ( B ) cultured for 48 h with IL-1β or medium alone. Numbers in contour plots show the percentage of cells in each quadrant. Data are representative of three independent experiments.
    Figure Legend Snippet: IL-1β induces the expression of MHC class II and costimulatory molecules on peripheral NCR − ILC3s. Expression of CD69, CD40, CD80, CD86, and MHC class II on sort-purified in vitro-generated CD4 + NCR − ILC3s ( A ) and ex vivo-isolated splenic CD4 + NCR − ILC3s ( B ) cultured for 48 h with IL-1β or medium alone. Numbers in contour plots show the percentage of cells in each quadrant. Data are representative of three independent experiments.

    Techniques Used: Expressing, Purification, In Vitro, Generated, Ex Vivo, Isolation, Cell Culture

    2) Product Images from "TRAF2 deficiency in B cells impairs CD40-induced isotype-switching that can be rescued by restoring NF-κB1 activation"

    Article Title: TRAF2 deficiency in B cells impairs CD40-induced isotype-switching that can be rescued by restoring NF-κB1 activation

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

    doi: 10.4049/jimmunol.1800337

    B cell-intrinsic TRAF2 but not TRAF3 deficiency impairs CD40-induced CSR ex vivo .
    Figure Legend Snippet: B cell-intrinsic TRAF2 but not TRAF3 deficiency impairs CD40-induced CSR ex vivo .

    Techniques Used: Ex Vivo

    PMA rescues the defective CD40-induced NF-κB1 complex activation and CSR in B-TRAF2-KO B cells.
    Figure Legend Snippet: PMA rescues the defective CD40-induced NF-κB1 complex activation and CSR in B-TRAF2-KO B cells.

    Techniques Used: Activation Assay

    Effects of B cell-intrinsic TRAF2 or TRAF3 deficiency on AID expression and GLT of Cγ1 or Cε induced by CD40.
    Figure Legend Snippet: Effects of B cell-intrinsic TRAF2 or TRAF3 deficiency on AID expression and GLT of Cγ1 or Cε induced by CD40.

    Techniques Used: Expressing

    TRAF2 is required for CD40 signaling to activate the NF-κB1 complex and induce isotype-switching.
    Figure Legend Snippet: TRAF2 is required for CD40 signaling to activate the NF-κB1 complex and induce isotype-switching.

    Techniques Used:

    3) Product Images from "T Follicular Helper-Like Cells Are Involved in the Pathogenesis of Experimental Autoimmune Encephalomyelitis"

    Article Title: T Follicular Helper-Like Cells Are Involved in the Pathogenesis of Experimental Autoimmune Encephalomyelitis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00944

    T follicular helper (Tfh)-like cells help B cells product autoantibody in an IL-21 and CD40 ligand (CD40L)-dependent manner. (A) Tfh-like and B cells from healthy controls (HCs) and experimental autoimmune encephalomyelitis (EAE) mice were cocultured. The level of anti-MOG 35–55 antibodies were measured by chemiluminescent enzyme-linked immunosorbent assay (CLISA) in the supernatant of Tfh/B coculturing system. H Tfh-like and H B stand for Tfh-like from HCs and B cells from HCs, respectively. E Tfh-like and E B stand for Tfh-like from EAE mice and B cells from EAE mice, respectively. *** P
    Figure Legend Snippet: T follicular helper (Tfh)-like cells help B cells product autoantibody in an IL-21 and CD40 ligand (CD40L)-dependent manner. (A) Tfh-like and B cells from healthy controls (HCs) and experimental autoimmune encephalomyelitis (EAE) mice were cocultured. The level of anti-MOG 35–55 antibodies were measured by chemiluminescent enzyme-linked immunosorbent assay (CLISA) in the supernatant of Tfh/B coculturing system. H Tfh-like and H B stand for Tfh-like from HCs and B cells from HCs, respectively. E Tfh-like and E B stand for Tfh-like from EAE mice and B cells from EAE mice, respectively. *** P

    Techniques Used: Mouse Assay, Chemiluminescent ELISA

    Schematic diagram of interaction between T follicular helper (Tfh) and B cells initiating synergetic effect of IL-21/IL-21R and CD40 ligand (CD40L)/CD40 to promote plasma cell differentiation and antibody production in EAE. (A) The process of antigen-activated T cells and B cells migrating to the B cell follicles, where enables B cells receive helper signals from Tfh cells. Red pentacle cells stand for dentritic cells and green pentacle ones for follicular dentritic cells. (B) At the T-B border, B cells receive signals from cognate CD4 + T cells to activate and proliferate. (C) Within the germinal centers (GCs), MOG-specific B cells recognize antigen and present it to Tfh cells. After receiving activation signal of MHC-peptide complex and ICOSL from B cells, Tfh cells secret large amount of IL-21 and express CD40L. IL-21 and CD40L bind to the relevant receptor and ligand on B cells, respectively. The ligation of CD40L and CD40 promote the stabilization of NF-κB-inducing kinase (NIK), and activate the downstream pathway, which lead the formation of P52 and initiate the expression of B lymphocyte-induced maturation protein-1 (Blimp-1). IL-21 receptor activates the JAK/STAT3 pathway and phosphorylated STAT3 (p-STAT3) promotes the expression of Blimp-1. Moreover, P-STAT3 could also upregulate the expression of NIK, probably through stimulating the de novo synthesis of NIK (red thin arrow). Thus, the signal provided by IL-21 and CD40L synergistically facilitates the differentiation of B cells and autoantibody production.
    Figure Legend Snippet: Schematic diagram of interaction between T follicular helper (Tfh) and B cells initiating synergetic effect of IL-21/IL-21R and CD40 ligand (CD40L)/CD40 to promote plasma cell differentiation and antibody production in EAE. (A) The process of antigen-activated T cells and B cells migrating to the B cell follicles, where enables B cells receive helper signals from Tfh cells. Red pentacle cells stand for dentritic cells and green pentacle ones for follicular dentritic cells. (B) At the T-B border, B cells receive signals from cognate CD4 + T cells to activate and proliferate. (C) Within the germinal centers (GCs), MOG-specific B cells recognize antigen and present it to Tfh cells. After receiving activation signal of MHC-peptide complex and ICOSL from B cells, Tfh cells secret large amount of IL-21 and express CD40L. IL-21 and CD40L bind to the relevant receptor and ligand on B cells, respectively. The ligation of CD40L and CD40 promote the stabilization of NF-κB-inducing kinase (NIK), and activate the downstream pathway, which lead the formation of P52 and initiate the expression of B lymphocyte-induced maturation protein-1 (Blimp-1). IL-21 receptor activates the JAK/STAT3 pathway and phosphorylated STAT3 (p-STAT3) promotes the expression of Blimp-1. Moreover, P-STAT3 could also upregulate the expression of NIK, probably through stimulating the de novo synthesis of NIK (red thin arrow). Thus, the signal provided by IL-21 and CD40L synergistically facilitates the differentiation of B cells and autoantibody production.

    Techniques Used: Cell Differentiation, Activation Assay, Ligation, Expressing

    4) Product Images from "T Follicular Helper-Like Cells Are Involved in the Pathogenesis of Experimental Autoimmune Encephalomyelitis"

    Article Title: T Follicular Helper-Like Cells Are Involved in the Pathogenesis of Experimental Autoimmune Encephalomyelitis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00944

    T follicular helper (Tfh)-like cells help B cells product autoantibody in an IL-21 and CD40 ligand (CD40L)-dependent manner. (A) Tfh-like and B cells from healthy controls (HCs) and experimental autoimmune encephalomyelitis (EAE) mice were cocultured. The level of anti-MOG 35–55 antibodies were measured by chemiluminescent enzyme-linked immunosorbent assay (CLISA) in the supernatant of Tfh/B coculturing system. H Tfh-like and H B stand for Tfh-like from HCs and B cells from HCs, respectively. E Tfh-like and E B stand for Tfh-like from EAE mice and B cells from EAE mice, respectively. *** P
    Figure Legend Snippet: T follicular helper (Tfh)-like cells help B cells product autoantibody in an IL-21 and CD40 ligand (CD40L)-dependent manner. (A) Tfh-like and B cells from healthy controls (HCs) and experimental autoimmune encephalomyelitis (EAE) mice were cocultured. The level of anti-MOG 35–55 antibodies were measured by chemiluminescent enzyme-linked immunosorbent assay (CLISA) in the supernatant of Tfh/B coculturing system. H Tfh-like and H B stand for Tfh-like from HCs and B cells from HCs, respectively. E Tfh-like and E B stand for Tfh-like from EAE mice and B cells from EAE mice, respectively. *** P

    Techniques Used: Mouse Assay, Chemiluminescent ELISA

    Schematic diagram of interaction between T follicular helper (Tfh) and B cells initiating synergetic effect of IL-21/IL-21R and CD40 ligand (CD40L)/CD40 to promote plasma cell differentiation and antibody production in EAE. (A) The process of antigen-activated T cells and B cells migrating to the B cell follicles, where enables B cells receive helper signals from Tfh cells. Red pentacle cells stand for dentritic cells and green pentacle ones for follicular dentritic cells. (B) At the T-B border, B cells receive signals from cognate CD4 + T cells to activate and proliferate. (C) Within the germinal centers (GCs), MOG-specific B cells recognize antigen and present it to Tfh cells. After receiving activation signal of MHC-peptide complex and ICOSL from B cells, Tfh cells secret large amount of IL-21 and express CD40L. IL-21 and CD40L bind to the relevant receptor and ligand on B cells, respectively. The ligation of CD40L and CD40 promote the stabilization of NF-κB-inducing kinase (NIK), and activate the downstream pathway, which lead the formation of P52 and initiate the expression of B lymphocyte-induced maturation protein-1 (Blimp-1). IL-21 receptor activates the JAK/STAT3 pathway and phosphorylated STAT3 (p-STAT3) promotes the expression of Blimp-1. Moreover, P-STAT3 could also upregulate the expression of NIK, probably through stimulating the de novo synthesis of NIK (red thin arrow). Thus, the signal provided by IL-21 and CD40L synergistically facilitates the differentiation of B cells and autoantibody production.
    Figure Legend Snippet: Schematic diagram of interaction between T follicular helper (Tfh) and B cells initiating synergetic effect of IL-21/IL-21R and CD40 ligand (CD40L)/CD40 to promote plasma cell differentiation and antibody production in EAE. (A) The process of antigen-activated T cells and B cells migrating to the B cell follicles, where enables B cells receive helper signals from Tfh cells. Red pentacle cells stand for dentritic cells and green pentacle ones for follicular dentritic cells. (B) At the T-B border, B cells receive signals from cognate CD4 + T cells to activate and proliferate. (C) Within the germinal centers (GCs), MOG-specific B cells recognize antigen and present it to Tfh cells. After receiving activation signal of MHC-peptide complex and ICOSL from B cells, Tfh cells secret large amount of IL-21 and express CD40L. IL-21 and CD40L bind to the relevant receptor and ligand on B cells, respectively. The ligation of CD40L and CD40 promote the stabilization of NF-κB-inducing kinase (NIK), and activate the downstream pathway, which lead the formation of P52 and initiate the expression of B lymphocyte-induced maturation protein-1 (Blimp-1). IL-21 receptor activates the JAK/STAT3 pathway and phosphorylated STAT3 (p-STAT3) promotes the expression of Blimp-1. Moreover, P-STAT3 could also upregulate the expression of NIK, probably through stimulating the de novo synthesis of NIK (red thin arrow). Thus, the signal provided by IL-21 and CD40L synergistically facilitates the differentiation of B cells and autoantibody production.

    Techniques Used: Cell Differentiation, Activation Assay, Ligation, Expressing

    5) Product Images from "Unique properties of thymic antigen-presenting cells promote epigenetic imprinting of alloantigen-specific regulatory T cells"

    Article Title: Unique properties of thymic antigen-presenting cells promote epigenetic imprinting of alloantigen-specific regulatory T cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.16221

    Expression of costimulatory molecules by thymic APCs APCs were enriched from thymi and spleens of BALB/c mice by enzymatic digestion and gradient centrifugation. Expression of CD86, CD70, CD83, CD137L, OX40L and CD40 was analyzed on gated CD45 − EpCAM + Ly51 − mTECs, CD45 + CD11c hi Lin − t-DCs and CD11c hi Lin − sp-DCs (Lin defined as CD90, CD49b, F4/80 and CD19) by flow cytometry. Gated CD4SP-Foxp3 − thymocytes were taken as control. Representative histograms from one out of four (CD86 and CD70), five (CD40) or three (CD83, CD137L and OX40L) independent experiments are depicted.
    Figure Legend Snippet: Expression of costimulatory molecules by thymic APCs APCs were enriched from thymi and spleens of BALB/c mice by enzymatic digestion and gradient centrifugation. Expression of CD86, CD70, CD83, CD137L, OX40L and CD40 was analyzed on gated CD45 − EpCAM + Ly51 − mTECs, CD45 + CD11c hi Lin − t-DCs and CD11c hi Lin − sp-DCs (Lin defined as CD90, CD49b, F4/80 and CD19) by flow cytometry. Gated CD4SP-Foxp3 − thymocytes were taken as control. Representative histograms from one out of four (CD86 and CD70), five (CD40) or three (CD83, CD137L and OX40L) independent experiments are depicted.

    Techniques Used: Expressing, Mouse Assay, Gradient Centrifugation, Flow Cytometry, Cytometry

    CD40-CD40L signaling is not critically required for generation of stable allo-iTregs A. Thymocytes and splenocytes from CD11c Cre xCD40 fl/fl (DC-specific CD40 knockout, filled circles) and CD11c WT xCD40 fl/fl mice (CD40 competent control, open circles) were analyzed by flow cytometry. Graphs show frequency of CD4SP thymocytes, Foxp3 + cells among CD4SP thymocytes, CD4 + CD3 + splenocytes and Foxp3 + Tregs cells among CD4 + CD3 + splenocytes. Data are summarized from two independent experiments (mean ± SD) and tested for significance using Mann-Whitney test; * p
    Figure Legend Snippet: CD40-CD40L signaling is not critically required for generation of stable allo-iTregs A. Thymocytes and splenocytes from CD11c Cre xCD40 fl/fl (DC-specific CD40 knockout, filled circles) and CD11c WT xCD40 fl/fl mice (CD40 competent control, open circles) were analyzed by flow cytometry. Graphs show frequency of CD4SP thymocytes, Foxp3 + cells among CD4SP thymocytes, CD4 + CD3 + splenocytes and Foxp3 + Tregs cells among CD4 + CD3 + splenocytes. Data are summarized from two independent experiments (mean ± SD) and tested for significance using Mann-Whitney test; * p

    Techniques Used: Knock-Out, Mouse Assay, Flow Cytometry, Cytometry, MANN-WHITNEY

    6) Product Images from "TRAF2 deficiency in B cells impairs CD40-induced isotype-switching that can be rescued by restoring NF-κB1 activation"

    Article Title: TRAF2 deficiency in B cells impairs CD40-induced isotype-switching that can be rescued by restoring NF-κB1 activation

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

    doi: 10.4049/jimmunol.1800337

    B cell-intrinsic TRAF2 but not TRAF3 deficiency impairs CD40-induced CSR ex vivo .
    Figure Legend Snippet: B cell-intrinsic TRAF2 but not TRAF3 deficiency impairs CD40-induced CSR ex vivo .

    Techniques Used: Ex Vivo

    PMA rescues the defective CD40-induced NF-κB1 complex activation and CSR in B-TRAF2-KO B cells.
    Figure Legend Snippet: PMA rescues the defective CD40-induced NF-κB1 complex activation and CSR in B-TRAF2-KO B cells.

    Techniques Used: Activation Assay

    Effects of B cell-intrinsic TRAF2 or TRAF3 deficiency on AID expression and GLT of Cγ1 or Cε induced by CD40.
    Figure Legend Snippet: Effects of B cell-intrinsic TRAF2 or TRAF3 deficiency on AID expression and GLT of Cγ1 or Cε induced by CD40.

    Techniques Used: Expressing

    TRAF2 is required for CD40 signaling to activate the NF-κB1 complex and induce isotype-switching.
    Figure Legend Snippet: TRAF2 is required for CD40 signaling to activate the NF-κB1 complex and induce isotype-switching.

    Techniques Used:

    7) Product Images from "Relevance of PSGL-1 Expression in B Cell Development and Activation"

    Article Title: Relevance of PSGL-1 Expression in B Cell Development and Activation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.588212

    PSGL-1 signaling inhibits in vitro activation of human peripheral B cells. (A) Relative frequencies of the different immunoglobulin (Ig)-expressing cell subsets in human total B cells (CD19 + ) and in the CD27 + and CD27 − B cell subpopulations. (B) Percentage of PSGL-1 + cells in the different Ig- expressing subsets of CD27 + and CD27 − B cells. (C) Relative frequencies of the different Ig- expressing cell subsets in the PSGL-1 + B cell subpopulation. (D) Percentage of surviving B cells after 72 h in culture: non-stimulated, activated with anti-IgM+anti-CD40 antibodies or activated with anti-IgM+anti-CD40+anti-PSGL-1 antibodies. (E) Relative frequency of CD27 + and CD27 − B cells in the total B cell population after 72 h in culture: non-stimulated, activated with anti-IgM+anti-CD40 antibodies or activated with anti-IgM+anti-CD40+anti-PSGL-1 antibodies. (F, G) Percentage of IL-10 + (F) and IgG + (G) cells in CD27 + and CD27 − B cells after 72 h in culture: non-stimulated, activated with anti-IgM+anti-CD40 antibodies or activated with anti-IgM+anti-CD40+anti-PSGL-1 antibodies. Bars represent the mean+standard deviation. *p
    Figure Legend Snippet: PSGL-1 signaling inhibits in vitro activation of human peripheral B cells. (A) Relative frequencies of the different immunoglobulin (Ig)-expressing cell subsets in human total B cells (CD19 + ) and in the CD27 + and CD27 − B cell subpopulations. (B) Percentage of PSGL-1 + cells in the different Ig- expressing subsets of CD27 + and CD27 − B cells. (C) Relative frequencies of the different Ig- expressing cell subsets in the PSGL-1 + B cell subpopulation. (D) Percentage of surviving B cells after 72 h in culture: non-stimulated, activated with anti-IgM+anti-CD40 antibodies or activated with anti-IgM+anti-CD40+anti-PSGL-1 antibodies. (E) Relative frequency of CD27 + and CD27 − B cells in the total B cell population after 72 h in culture: non-stimulated, activated with anti-IgM+anti-CD40 antibodies or activated with anti-IgM+anti-CD40+anti-PSGL-1 antibodies. (F, G) Percentage of IL-10 + (F) and IgG + (G) cells in CD27 + and CD27 − B cells after 72 h in culture: non-stimulated, activated with anti-IgM+anti-CD40 antibodies or activated with anti-IgM+anti-CD40+anti-PSGL-1 antibodies. Bars represent the mean+standard deviation. *p

    Techniques Used: In Vitro, Activation Assay, Expressing, Standard Deviation

    8) Product Images from "TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer"

    Article Title: TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer

    Journal: Science immunology

    doi: 10.1126/sciimmunol.aau7523

    TET proteins facilitate CSR. ( A ) Flowchart of in vivo experiment. f.p., footpad. ( B ) Top: Flow cytometric analysis of CD19 + GL7 + Fas + GC B cells at the draining popliteal lymph nodes from WT and Tet2/3 DKO mice after NP-OVA immunization as in (A). Bottom: Decreased IgG1-switched cells among GC B cells in Tet2/3 DKO (YFP + GC B–gated) compared with WT mice (GC B–gated). ( C to F ) Quantification of experiments shown in (B). Data shown are aggregated results from two independent experiments. Means and SEs are shown. WT, n = 11; DKO, n = 12. ( G ) Flowchart of in vitro IgG1 switching. Cells were labeled with Cell trace violet and activated for 4 days with LPS and IL-4. ( H and I ) Flow cytometry plots (H) and quantification (I) of IgG1-switched B cells in Tet2/3 DKO ( n = 4) and WT ( n = 4) mice. Representative of at least three independent experiments. ( J ) Circular γ1 transcript was quantified by qRT-PCR and normalized to Gapdh and then to the level of WT. Representative experiment of two independent experiments with three technical replicates. ( K ) Flowchart of in vitro IgA switching. Cells were activated for 5 days with anti-CD40, rmIL-4, rmIL-5, and rhTGFβ. ( L to N ) Flow cytometry plots (L) and quantification (M and N) of IgG1-switched (M) and IgA-switched cells (N). Representative of three independent experiments with three technical replicates. Statistical significance was calculated using unpaired two-tailed t test. * P
    Figure Legend Snippet: TET proteins facilitate CSR. ( A ) Flowchart of in vivo experiment. f.p., footpad. ( B ) Top: Flow cytometric analysis of CD19 + GL7 + Fas + GC B cells at the draining popliteal lymph nodes from WT and Tet2/3 DKO mice after NP-OVA immunization as in (A). Bottom: Decreased IgG1-switched cells among GC B cells in Tet2/3 DKO (YFP + GC B–gated) compared with WT mice (GC B–gated). ( C to F ) Quantification of experiments shown in (B). Data shown are aggregated results from two independent experiments. Means and SEs are shown. WT, n = 11; DKO, n = 12. ( G ) Flowchart of in vitro IgG1 switching. Cells were labeled with Cell trace violet and activated for 4 days with LPS and IL-4. ( H and I ) Flow cytometry plots (H) and quantification (I) of IgG1-switched B cells in Tet2/3 DKO ( n = 4) and WT ( n = 4) mice. Representative of at least three independent experiments. ( J ) Circular γ1 transcript was quantified by qRT-PCR and normalized to Gapdh and then to the level of WT. Representative experiment of two independent experiments with three technical replicates. ( K ) Flowchart of in vitro IgA switching. Cells were activated for 5 days with anti-CD40, rmIL-4, rmIL-5, and rhTGFβ. ( L to N ) Flow cytometry plots (L) and quantification (M and N) of IgG1-switched (M) and IgA-switched cells (N). Representative of three independent experiments with three technical replicates. Statistical significance was calculated using unpaired two-tailed t test. * P

    Techniques Used: In Vivo, Flow Cytometry, Mouse Assay, In Vitro, Labeling, Cytometry, Quantitative RT-PCR, Two Tailed Test

    9) Product Images from "Neutrophils Deficient in Innate Suppressor IRAK-M Enhances Anti-tumor Immune Responses"

    Article Title: Neutrophils Deficient in Innate Suppressor IRAK-M Enhances Anti-tumor Immune Responses

    Journal: Molecular Therapy

    doi: 10.1016/j.ymthe.2019.09.019

    IRAK-M Deficiency Releases the Neutrophil Suppression on the Proliferation and Activation of T Cells (A) CD80, CD40, and PD-L1 expression on GM-CSF primed WT or IRAK-M −/− neutrophils. (B) To monitor T cell proliferation, CFSE-labeled T cells were co-cultured with GM-CSF primed WT or IRAK-M −/− neutrophils in the anti-CD3 antibody-coated plates for 72 h. Representative results are shown. (C and D) To monitor T cell activation, PD-1, CD40L, CD62L, Foxp3, on CD4 T cells (C), as well as PD-1, granzyme B, IFNγ, and CD107α in CD8 T cells (D) were analyzed using flow cytometry. Data, mean ± SEM. Student’s t test. *p
    Figure Legend Snippet: IRAK-M Deficiency Releases the Neutrophil Suppression on the Proliferation and Activation of T Cells (A) CD80, CD40, and PD-L1 expression on GM-CSF primed WT or IRAK-M −/− neutrophils. (B) To monitor T cell proliferation, CFSE-labeled T cells were co-cultured with GM-CSF primed WT or IRAK-M −/− neutrophils in the anti-CD3 antibody-coated plates for 72 h. Representative results are shown. (C and D) To monitor T cell activation, PD-1, CD40L, CD62L, Foxp3, on CD4 T cells (C), as well as PD-1, granzyme B, IFNγ, and CD107α in CD8 T cells (D) were analyzed using flow cytometry. Data, mean ± SEM. Student’s t test. *p

    Techniques Used: Activation Assay, Expressing, Labeling, Cell Culture, Flow Cytometry

    IRAK-M Deficiency Mediates the Neutrophil Suppression on the Proliferation and Activation T Cells via Enhanced CD80/CD40 and Reduced PD-L To monitor T cell proliferation, CFSE-labeled T cells were co-cultured with GM-CSF primed neutrophils in the anti-CD3 antibody-coated plates for 72 h, without or with anti-CD80 antibody (A), anti-CD40 antibody (D), or anti-PD-L1 antibody (G). To monitor T cell activation, PD-1, CD40L, CD62L on CD4 T cells, as well as CD62L, PD-1, granzyme B, IFNγ, and CD107α in CD8 T cells were analyzed using flow cytometry. (B and C) In the presence of anti-CD80 antibody, CD62L, CD40L on CD4 T cells (B), and CD62L and CD107α on CD8 + cells (C) were analyzed by flow cytometry. (E and F) In the presence of anti-CD40 antibody, CD62L, CD40L on CD4 T cells (E), and CD62L on CD8 T cells (F) were analyzed by flow cytometry. (H and I) In the presence of anti-PD-L1 antibody, CD62L, PD-1 on CD4 T cells (H), and PD-1 and granzyme B on CD8 T (I) cells were analyzed by flow cytometry. (J) Immunoblotting analysis of p-STAT1, p-STAT3, STAT3, p-STAT5, STAT5, and GAPDH in lysates from bone marrow neutrophils primed with or without GM-CSF overnight. (K) Schematic diagram of the role and regulation of IRAK-M in neutrophils with T cell communication. Data, mean ± SEM. Student’s t test. *p
    Figure Legend Snippet: IRAK-M Deficiency Mediates the Neutrophil Suppression on the Proliferation and Activation T Cells via Enhanced CD80/CD40 and Reduced PD-L To monitor T cell proliferation, CFSE-labeled T cells were co-cultured with GM-CSF primed neutrophils in the anti-CD3 antibody-coated plates for 72 h, without or with anti-CD80 antibody (A), anti-CD40 antibody (D), or anti-PD-L1 antibody (G). To monitor T cell activation, PD-1, CD40L, CD62L on CD4 T cells, as well as CD62L, PD-1, granzyme B, IFNγ, and CD107α in CD8 T cells were analyzed using flow cytometry. (B and C) In the presence of anti-CD80 antibody, CD62L, CD40L on CD4 T cells (B), and CD62L and CD107α on CD8 + cells (C) were analyzed by flow cytometry. (E and F) In the presence of anti-CD40 antibody, CD62L, CD40L on CD4 T cells (E), and CD62L on CD8 T cells (F) were analyzed by flow cytometry. (H and I) In the presence of anti-PD-L1 antibody, CD62L, PD-1 on CD4 T cells (H), and PD-1 and granzyme B on CD8 T (I) cells were analyzed by flow cytometry. (J) Immunoblotting analysis of p-STAT1, p-STAT3, STAT3, p-STAT5, STAT5, and GAPDH in lysates from bone marrow neutrophils primed with or without GM-CSF overnight. (K) Schematic diagram of the role and regulation of IRAK-M in neutrophils with T cell communication. Data, mean ± SEM. Student’s t test. *p

    Techniques Used: Activation Assay, Labeling, Cell Culture, Flow Cytometry

    Enhanced CD80/CD40 Expression and Reduced Expression of PD-L1 and CD11b on IRAK-M −/− Neutrophils (A and B) Percentages of neutrophils (Ly6G + CD11b + ) in spleen (A) and blood (B) from naive mice or mice with AOM/DSS treatment. (C and D) CD80, PD-L1, CD40, CD11b, and LRRC32 expression on spleen neutrophils from naive mice (C) or mice with AOM/DSS treatment (D). (E and F) CD80, PD-L1, CD40, and CD11b expression on blood neutrophils from naive mice (E) or mice with AOM/DSS treatment (F). Data, mean ± SEM. Student’s t test (C and G). *p
    Figure Legend Snippet: Enhanced CD80/CD40 Expression and Reduced Expression of PD-L1 and CD11b on IRAK-M −/− Neutrophils (A and B) Percentages of neutrophils (Ly6G + CD11b + ) in spleen (A) and blood (B) from naive mice or mice with AOM/DSS treatment. (C and D) CD80, PD-L1, CD40, CD11b, and LRRC32 expression on spleen neutrophils from naive mice (C) or mice with AOM/DSS treatment (D). (E and F) CD80, PD-L1, CD40, and CD11b expression on blood neutrophils from naive mice (E) or mice with AOM/DSS treatment (F). Data, mean ± SEM. Student’s t test (C and G). *p

    Techniques Used: Expressing, Mouse Assay

    10) Product Images from "TRAF2 deficiency in B cells impairs CD40-induced isotype-switching that can be rescued by restoring NF-κB1 activation"

    Article Title: TRAF2 deficiency in B cells impairs CD40-induced isotype-switching that can be rescued by restoring NF-κB1 activation

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

    doi: 10.4049/jimmunol.1800337

    B cell-intrinsic TRAF2 but not TRAF3 deficiency impairs CD40-induced CSR ex vivo .
    Figure Legend Snippet: B cell-intrinsic TRAF2 but not TRAF3 deficiency impairs CD40-induced CSR ex vivo .

    Techniques Used: Ex Vivo

    PMA rescues the defective CD40-induced NF-κB1 complex activation and CSR in B-TRAF2-KO B cells.
    Figure Legend Snippet: PMA rescues the defective CD40-induced NF-κB1 complex activation and CSR in B-TRAF2-KO B cells.

    Techniques Used: Activation Assay

    Effects of B cell-intrinsic TRAF2 or TRAF3 deficiency on AID expression and GLT of Cγ1 or Cε induced by CD40.
    Figure Legend Snippet: Effects of B cell-intrinsic TRAF2 or TRAF3 deficiency on AID expression and GLT of Cγ1 or Cε induced by CD40.

    Techniques Used: Expressing

    TRAF2 is required for CD40 signaling to activate the NF-κB1 complex and induce isotype-switching.
    Figure Legend Snippet: TRAF2 is required for CD40 signaling to activate the NF-κB1 complex and induce isotype-switching.

    Techniques Used:

    11) Product Images from "B Cell-Based Vaccine Transduced With ESAT6-Expressing Vaccinia Virus and Presenting α-Galactosylceramide Is a Novel Vaccine Candidate Against ESAT6-Expressing Mycobacterial Diseases"

    Article Title: B Cell-Based Vaccine Transduced With ESAT6-Expressing Vaccinia Virus and Presenting α-Galactosylceramide Is a Novel Vaccine Candidate Against ESAT6-Expressing Mycobacterial Diseases

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02542

    B/αGC/vacESAT6 up-regulates co-stimulatory molecules on B cells. (A,B) Vero cells were transduced with vaccinia-ESAT6 (vacESAT6) at a multiplicity of infection (MOI) of 1. Transduced cells were fluorescently stained for ESAT6 (green) and counterstained with DAPI (blue) for nuclei which were analyzed by confocal microscopy to detect the expression of ESAT6 (scale bar = 20 μm). (A) Representative confocal images and (B) Representation of the evaluation of green fluorescent area. (C,D) B220 + cells were isolated from splenocytes of naïve C57BL/6 mice. Isolated B220 + cells were transduced with vacESAT6 at a MOI of 1 and/or loaded with 1 μg/ml of αGC and then co-cultured with naïve splenocytes for 24 h. Incubated cells were stained to examine the expression of B220, CD40, and CD86. Expression levels of CD40 and CD86 in B220 + cells were analyzed by flow cytometry. (C) Representative flow cytometry histogram and (D) summary of mean fluorescence intensity of CD40 and CD86 expression in B cells. ANOVA. * p
    Figure Legend Snippet: B/αGC/vacESAT6 up-regulates co-stimulatory molecules on B cells. (A,B) Vero cells were transduced with vaccinia-ESAT6 (vacESAT6) at a multiplicity of infection (MOI) of 1. Transduced cells were fluorescently stained for ESAT6 (green) and counterstained with DAPI (blue) for nuclei which were analyzed by confocal microscopy to detect the expression of ESAT6 (scale bar = 20 μm). (A) Representative confocal images and (B) Representation of the evaluation of green fluorescent area. (C,D) B220 + cells were isolated from splenocytes of naïve C57BL/6 mice. Isolated B220 + cells were transduced with vacESAT6 at a MOI of 1 and/or loaded with 1 μg/ml of αGC and then co-cultured with naïve splenocytes for 24 h. Incubated cells were stained to examine the expression of B220, CD40, and CD86. Expression levels of CD40 and CD86 in B220 + cells were analyzed by flow cytometry. (C) Representative flow cytometry histogram and (D) summary of mean fluorescence intensity of CD40 and CD86 expression in B cells. ANOVA. * p

    Techniques Used: Transduction, Infection, Staining, Confocal Microscopy, Expressing, Isolation, Mouse Assay, Cell Culture, Incubation, Flow Cytometry, Cytometry, Fluorescence

    12) Product Images from "Proinflammatory T helper type 17 cells are effective B-cell helpers"

    Article Title: Proinflammatory T helper type 17 cells are effective B-cell helpers

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

    doi: 10.1073/pnas.1009234107

    IL-17 induces class switching in vitro. B cells were purified and activated in the presence of increasing doses of the indicated cytokines. Antibody production and class switch recombination were analyzed in response to stimulation with anti-CD40 (5 μg/mL)/IgM
    Figure Legend Snippet: IL-17 induces class switching in vitro. B cells were purified and activated in the presence of increasing doses of the indicated cytokines. Antibody production and class switch recombination were analyzed in response to stimulation with anti-CD40 (5 μg/mL)/IgM

    Techniques Used: In Vitro, Purification

    13) Product Images from "T Follicular Helper-Like Cells Are Involved in the Pathogenesis of Experimental Autoimmune Encephalomyelitis"

    Article Title: T Follicular Helper-Like Cells Are Involved in the Pathogenesis of Experimental Autoimmune Encephalomyelitis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00944

    T follicular helper (Tfh)-like cells help B cells product autoantibody in an IL-21 and CD40 ligand (CD40L)-dependent manner. (A) Tfh-like and B cells from healthy controls (HCs) and experimental autoimmune encephalomyelitis (EAE) mice were cocultured. The level of anti-MOG 35–55 antibodies were measured by chemiluminescent enzyme-linked immunosorbent assay (CLISA) in the supernatant of Tfh/B coculturing system. H Tfh-like and H B stand for Tfh-like from HCs and B cells from HCs, respectively. E Tfh-like and E B stand for Tfh-like from EAE mice and B cells from EAE mice, respectively. *** P
    Figure Legend Snippet: T follicular helper (Tfh)-like cells help B cells product autoantibody in an IL-21 and CD40 ligand (CD40L)-dependent manner. (A) Tfh-like and B cells from healthy controls (HCs) and experimental autoimmune encephalomyelitis (EAE) mice were cocultured. The level of anti-MOG 35–55 antibodies were measured by chemiluminescent enzyme-linked immunosorbent assay (CLISA) in the supernatant of Tfh/B coculturing system. H Tfh-like and H B stand for Tfh-like from HCs and B cells from HCs, respectively. E Tfh-like and E B stand for Tfh-like from EAE mice and B cells from EAE mice, respectively. *** P

    Techniques Used: Mouse Assay, Chemiluminescent ELISA

    Schematic diagram of interaction between T follicular helper (Tfh) and B cells initiating synergetic effect of IL-21/IL-21R and CD40 ligand (CD40L)/CD40 to promote plasma cell differentiation and antibody production in EAE. (A) The process of antigen-activated T cells and B cells migrating to the B cell follicles, where enables B cells receive helper signals from Tfh cells. Red pentacle cells stand for dentritic cells and green pentacle ones for follicular dentritic cells. (B) At the T-B border, B cells receive signals from cognate CD4 + T cells to activate and proliferate. (C) Within the germinal centers (GCs), MOG-specific B cells recognize antigen and present it to Tfh cells. After receiving activation signal of MHC-peptide complex and ICOSL from B cells, Tfh cells secret large amount of IL-21 and express CD40L. IL-21 and CD40L bind to the relevant receptor and ligand on B cells, respectively. The ligation of CD40L and CD40 promote the stabilization of NF-κB-inducing kinase (NIK), and activate the downstream pathway, which lead the formation of P52 and initiate the expression of B lymphocyte-induced maturation protein-1 (Blimp-1). IL-21 receptor activates the JAK/STAT3 pathway and phosphorylated STAT3 (p-STAT3) promotes the expression of Blimp-1. Moreover, P-STAT3 could also upregulate the expression of NIK, probably through stimulating the de novo synthesis of NIK (red thin arrow). Thus, the signal provided by IL-21 and CD40L synergistically facilitates the differentiation of B cells and autoantibody production.
    Figure Legend Snippet: Schematic diagram of interaction between T follicular helper (Tfh) and B cells initiating synergetic effect of IL-21/IL-21R and CD40 ligand (CD40L)/CD40 to promote plasma cell differentiation and antibody production in EAE. (A) The process of antigen-activated T cells and B cells migrating to the B cell follicles, where enables B cells receive helper signals from Tfh cells. Red pentacle cells stand for dentritic cells and green pentacle ones for follicular dentritic cells. (B) At the T-B border, B cells receive signals from cognate CD4 + T cells to activate and proliferate. (C) Within the germinal centers (GCs), MOG-specific B cells recognize antigen and present it to Tfh cells. After receiving activation signal of MHC-peptide complex and ICOSL from B cells, Tfh cells secret large amount of IL-21 and express CD40L. IL-21 and CD40L bind to the relevant receptor and ligand on B cells, respectively. The ligation of CD40L and CD40 promote the stabilization of NF-κB-inducing kinase (NIK), and activate the downstream pathway, which lead the formation of P52 and initiate the expression of B lymphocyte-induced maturation protein-1 (Blimp-1). IL-21 receptor activates the JAK/STAT3 pathway and phosphorylated STAT3 (p-STAT3) promotes the expression of Blimp-1. Moreover, P-STAT3 could also upregulate the expression of NIK, probably through stimulating the de novo synthesis of NIK (red thin arrow). Thus, the signal provided by IL-21 and CD40L synergistically facilitates the differentiation of B cells and autoantibody production.

    Techniques Used: Cell Differentiation, Activation Assay, Ligation, Expressing

    14) Product Images from "TRAF2 deficiency in B cells impairs CD40-induced isotype-switching that can be rescued by restoring NF-κB1 activation"

    Article Title: TRAF2 deficiency in B cells impairs CD40-induced isotype-switching that can be rescued by restoring NF-κB1 activation

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

    doi: 10.4049/jimmunol.1800337

    B cell-intrinsic TRAF2 but not TRAF3 deficiency impairs CD40-induced CSR ex vivo .
    Figure Legend Snippet: B cell-intrinsic TRAF2 but not TRAF3 deficiency impairs CD40-induced CSR ex vivo .

    Techniques Used: Ex Vivo

    PMA rescues the defective CD40-induced NF-κB1 complex activation and CSR in B-TRAF2-KO B cells.
    Figure Legend Snippet: PMA rescues the defective CD40-induced NF-κB1 complex activation and CSR in B-TRAF2-KO B cells.

    Techniques Used: Activation Assay

    Effects of B cell-intrinsic TRAF2 or TRAF3 deficiency on AID expression and GLT of Cγ1 or Cε induced by CD40.
    Figure Legend Snippet: Effects of B cell-intrinsic TRAF2 or TRAF3 deficiency on AID expression and GLT of Cγ1 or Cε induced by CD40.

    Techniques Used: Expressing

    TRAF2 is required for CD40 signaling to activate the NF-κB1 complex and induce isotype-switching.
    Figure Legend Snippet: TRAF2 is required for CD40 signaling to activate the NF-κB1 complex and induce isotype-switching.

    Techniques Used:

    15) Product Images from "HMCES functions in the alternative end-joining pathway of the DNA DSB repair during class switch recombination in B cells."

    Article Title: HMCES functions in the alternative end-joining pathway of the DNA DSB repair during class switch recombination in B cells.

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2019.10.031

    HMCES deficiency results in defects in DNA DSB repair through the microhomology-mediated alternative end-joining pathway. A) Bar graphs quantifying the distribution of observed microhomologies in Sμ-Sα switch junctions of WT CH12, ΔHMCES CH12 clones H3 and H23, and ΔHMCES CH12 clone H3 reconstituted with FL HMCES protein (detected by PCR amplification and Sanger sequencing). Data are representative of two independent experiments. Statistical significance was computed using Welch’s T-test. B) Pie charts displaying distribution of Sμ-Sα switch region microhomologies of 0–3 or > 3 nucleotides in WT CH12, ΔHMCES CH12 clones H3 and H23, and ΔHMCES CH12 clone H3 reconstituted with FL HMCES. Cells were stimulated with anti-CD40, Interleukin-4 (IL-4) and Transforming growth factor-β (TGF-β) for 72 hours. C) Bar graphs quantifying the distribution of observed microhomologies in Sμ-Sγ1 switch junctions of Hmces +/+ and Hmces −/− primary B cells (detected by PCR amplification and Sanger sequencing) stimulated with anti-CD40 and IL-4 for 4 days. Data are representative of two independent experiments. Statistical significance was computed using Welch’s T-test. D) Pie charts displaying distribution of Sμ-Sγ1 switch junctional microhomologies of 0–3 or > 3 nucleotides in Hmces +/+ and Hmces −/− primary B cells stimulated with anti-CD40 and IL-4 for 4 days. E) Bar graphs showing the relative percentage of GFP-positive cells in ΔHMCES U2OS-EJ2-Alt-EJ reporter cells ( left ) or ΔHMCES U2OS-EJ5-cNHEJ reporter cells ( right ) compared with corresponding WT U2OS reporter cells. Data are represented as mean ± SD and are representative of at least three independent experiments. Statistical significance was calculated using student t-test. **p value ≤0.0001, *p value ≤0.0005.
    Figure Legend Snippet: HMCES deficiency results in defects in DNA DSB repair through the microhomology-mediated alternative end-joining pathway. A) Bar graphs quantifying the distribution of observed microhomologies in Sμ-Sα switch junctions of WT CH12, ΔHMCES CH12 clones H3 and H23, and ΔHMCES CH12 clone H3 reconstituted with FL HMCES protein (detected by PCR amplification and Sanger sequencing). Data are representative of two independent experiments. Statistical significance was computed using Welch’s T-test. B) Pie charts displaying distribution of Sμ-Sα switch region microhomologies of 0–3 or > 3 nucleotides in WT CH12, ΔHMCES CH12 clones H3 and H23, and ΔHMCES CH12 clone H3 reconstituted with FL HMCES. Cells were stimulated with anti-CD40, Interleukin-4 (IL-4) and Transforming growth factor-β (TGF-β) for 72 hours. C) Bar graphs quantifying the distribution of observed microhomologies in Sμ-Sγ1 switch junctions of Hmces +/+ and Hmces −/− primary B cells (detected by PCR amplification and Sanger sequencing) stimulated with anti-CD40 and IL-4 for 4 days. Data are representative of two independent experiments. Statistical significance was computed using Welch’s T-test. D) Pie charts displaying distribution of Sμ-Sγ1 switch junctional microhomologies of 0–3 or > 3 nucleotides in Hmces +/+ and Hmces −/− primary B cells stimulated with anti-CD40 and IL-4 for 4 days. E) Bar graphs showing the relative percentage of GFP-positive cells in ΔHMCES U2OS-EJ2-Alt-EJ reporter cells ( left ) or ΔHMCES U2OS-EJ5-cNHEJ reporter cells ( right ) compared with corresponding WT U2OS reporter cells. Data are represented as mean ± SD and are representative of at least three independent experiments. Statistical significance was calculated using student t-test. **p value ≤0.0001, *p value ≤0.0005.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Amplification, Sequencing

    HMCES deficiency leads to a defect in CSR in the CH12 B cell line. A) Representative flow cytometry plots of WT or ΔHMCES CH12 cells (clones H3 and H23). Cells were stimulated with anti-CD40, Interleukin-4 (IL-4) and Transforming growth factor-β (TGF-β) (CIT) and CSR from IgM to IgA was measured after 72 hours. B) Bar graph showing the relative percent CSR in ΔHMCES CH12 clones H3 and H23 compared with WT CH12 cells after 72 hours of CIT stimulation. C) Flow cytometry plots showing the frequency of CSR to IgA after 72 hours of CIT stimulation in WT and ΔHMCES CH12 cells transduced with either empty lentiviral vector or vector expressing full length (FL) HMCES protein. The ΔHMCES plot is representative of both H3 and H23 ΔHMCES CH12 cells. D) Bar graphs showing the relative percent CSR in ΔHMCES CH12 cells, normalized to WT CH12 cells transduced with empty vector in the same experiment. Data from H3 and H23 ΔHMCES CH12 cells are merged. E) Diagram indicating selected known features of human HMCES protein tested with the mutational analysis. F) Flow cytometry plots showing the frequency of CSR to IgA after 72 hours of CIT stimulation in WT and ΔHMCES CH12 cells transduced with either empty lentiviral vector or vectors expressing HMCES 1–270 (SRAPd), HMCES C2A or HMCES R212A variants generated on FL HMCES backbone. The ΔHMCES plot is representative of both H3 and H23 ΔHMCES CH12 cells. G) Bar graph showing the relative percent CSR of WT or ΔHMCES (both H3 and H23) CH12 cells lentivirally transduced with empty vector, HMCES 1–270 (SRAPd), HMCES C2A and HMCES R212A variant proteins generated on. CSR is normalized to WT CH12 cells transduced with empty vector in the same experiment. Data are representative of 4 or more independent experiments with each dot representing an independent experiment. Data are represented as mean ± SD. Statistical significance was calculated using student t-test. ***p value ≤0.000001, *p value ≤0.001 (panels B and D) or by two-way ANOVA **p value ≤0.0001 (panel G).
    Figure Legend Snippet: HMCES deficiency leads to a defect in CSR in the CH12 B cell line. A) Representative flow cytometry plots of WT or ΔHMCES CH12 cells (clones H3 and H23). Cells were stimulated with anti-CD40, Interleukin-4 (IL-4) and Transforming growth factor-β (TGF-β) (CIT) and CSR from IgM to IgA was measured after 72 hours. B) Bar graph showing the relative percent CSR in ΔHMCES CH12 clones H3 and H23 compared with WT CH12 cells after 72 hours of CIT stimulation. C) Flow cytometry plots showing the frequency of CSR to IgA after 72 hours of CIT stimulation in WT and ΔHMCES CH12 cells transduced with either empty lentiviral vector or vector expressing full length (FL) HMCES protein. The ΔHMCES plot is representative of both H3 and H23 ΔHMCES CH12 cells. D) Bar graphs showing the relative percent CSR in ΔHMCES CH12 cells, normalized to WT CH12 cells transduced with empty vector in the same experiment. Data from H3 and H23 ΔHMCES CH12 cells are merged. E) Diagram indicating selected known features of human HMCES protein tested with the mutational analysis. F) Flow cytometry plots showing the frequency of CSR to IgA after 72 hours of CIT stimulation in WT and ΔHMCES CH12 cells transduced with either empty lentiviral vector or vectors expressing HMCES 1–270 (SRAPd), HMCES C2A or HMCES R212A variants generated on FL HMCES backbone. The ΔHMCES plot is representative of both H3 and H23 ΔHMCES CH12 cells. G) Bar graph showing the relative percent CSR of WT or ΔHMCES (both H3 and H23) CH12 cells lentivirally transduced with empty vector, HMCES 1–270 (SRAPd), HMCES C2A and HMCES R212A variant proteins generated on. CSR is normalized to WT CH12 cells transduced with empty vector in the same experiment. Data are representative of 4 or more independent experiments with each dot representing an independent experiment. Data are represented as mean ± SD. Statistical significance was calculated using student t-test. ***p value ≤0.000001, *p value ≤0.001 (panels B and D) or by two-way ANOVA **p value ≤0.0001 (panel G).

    Techniques Used: Flow Cytometry, Clone Assay, Transduction, Plasmid Preparation, Expressing, Generated, Variant Assay

    Genetic disruption of c-NHEJ in HMCES-deficient CH12 cells results in a striking decrease in DNA DSB repair. A) Representative flow cytometry plots showing CSR to IgA in CH12 shNT, CH12 shKU70, ΔHMCES shNT and ΔHMCES shKU70 cells stimulated with anti-CD40, Interleukin-4 (IL-4) and Transforming growth factor-β (TGF-β) (CIT) for 72 hours. B) Bar graphs quantifying the relative percent of CSR to IgA in WT or ΔHMCES (H23) CH12 cells lentivirally transduced with a non-targeting shRNA (shNT) or shRNA against KU70 (shKU70). Percent switching is normalized to WT CH12 cells transduced with shNT in each experiment. C) Representative flow cytometry plots showing CSR to IgA in WT, ΔHMCES (H3 and H23), ΔLIGASE4 (CH12 L12) and, ΔHMCES and LIGASE4 double-deficient CH12 cells (H3 L15, H23 L2, H23 L18) after 72 hours stimulation with CIT. D) Bar graphs quantifying the relative percent CSR to IgA in WT, ΔHMCES (H3 and H23), ΔLIGASE4 (CH12 L12) and, ΔHMCES and LIGASE4 double-deficient CH12 cells (H3 L15, H23 L2, H23 L18). E) Representative flow cytometry plots showing switching to IgA in CH12 shNT, CH12 shCTIP-1, ΔHMCES shNT, ΔHMCES shCTIP-1 and ΔHMCES shCTIP-2 cells stimulated with CIT for 72 hours. F) Bar graphs quantifying CSR to IgA in WT or ΔHMCES (H23) CH12 cells lentivirally transduced with a non-targeting shRNA (shNT) or shRNAs against CTIP (shCTIP-1 and shCTIP-2). Percent switching is normalized to WT CH12 cells transduced with shNT in each experiment. Data in panels B and D are represented as mean ± SD and each dot represents an independent experiment. Statistical significance was calculated using student t-test. *p value ≤0.0005, **p value ≤0.00001.
    Figure Legend Snippet: Genetic disruption of c-NHEJ in HMCES-deficient CH12 cells results in a striking decrease in DNA DSB repair. A) Representative flow cytometry plots showing CSR to IgA in CH12 shNT, CH12 shKU70, ΔHMCES shNT and ΔHMCES shKU70 cells stimulated with anti-CD40, Interleukin-4 (IL-4) and Transforming growth factor-β (TGF-β) (CIT) for 72 hours. B) Bar graphs quantifying the relative percent of CSR to IgA in WT or ΔHMCES (H23) CH12 cells lentivirally transduced with a non-targeting shRNA (shNT) or shRNA against KU70 (shKU70). Percent switching is normalized to WT CH12 cells transduced with shNT in each experiment. C) Representative flow cytometry plots showing CSR to IgA in WT, ΔHMCES (H3 and H23), ΔLIGASE4 (CH12 L12) and, ΔHMCES and LIGASE4 double-deficient CH12 cells (H3 L15, H23 L2, H23 L18) after 72 hours stimulation with CIT. D) Bar graphs quantifying the relative percent CSR to IgA in WT, ΔHMCES (H3 and H23), ΔLIGASE4 (CH12 L12) and, ΔHMCES and LIGASE4 double-deficient CH12 cells (H3 L15, H23 L2, H23 L18). E) Representative flow cytometry plots showing switching to IgA in CH12 shNT, CH12 shCTIP-1, ΔHMCES shNT, ΔHMCES shCTIP-1 and ΔHMCES shCTIP-2 cells stimulated with CIT for 72 hours. F) Bar graphs quantifying CSR to IgA in WT or ΔHMCES (H23) CH12 cells lentivirally transduced with a non-targeting shRNA (shNT) or shRNAs against CTIP (shCTIP-1 and shCTIP-2). Percent switching is normalized to WT CH12 cells transduced with shNT in each experiment. Data in panels B and D are represented as mean ± SD and each dot represents an independent experiment. Statistical significance was calculated using student t-test. *p value ≤0.0005, **p value ≤0.00001.

    Techniques Used: Non-Homologous End Joining, Flow Cytometry, Transduction, shRNA

    HMCES leads to a B cell intrinsic defect in CSR. A) Representative flow cytometry plots showing CSR to IgG1 and IgA in B cells isolated from Hmces +/+ and Hmces −/− mice and stimulated with anti-CD40, IL-4, TGF-β and IL-5 for 5 days. B) Bar graphs quantifying the kinetics of IgM to IgA switching in primary B cells from Hmces +/+ and Hmces −/− mice. C) Bar graphs quantifying the kinetics of IgM to IgG1 switching in primary B cells isolated from Hmces +/+ and Hmces −/− mice stimulated with anti-CD40, IL-4, TGF-β and IL-5. D) Bar graphs quantifying the kinetics of IgM to IgG1 switching in primary B cells isolated from Hmces +/+ and Hmces −/− mice and stimulated with anti-CD40 and IL-4. E) Bar graphs quantifying the kinetics of IgM to IgE switching in primary B cells isolated from Hmces +/+ and Hmces −/− mice and stimulated with anti-CD40 and IL-4. F) Bar graphs quantifying the kinetics of IgM to IgG3 switching in primary B cells isolated from Hmces +/+ and Hmces −/− mice and stimulated with LPS. Data are represented as mean ± SD. Each dot represents cells isolated from an individual mouse and lines are used to highlight samples analyzed in the same experiment. Statistical significance was calculated using two-way ANOVA. *p value ≤0.005, **p value ≤0.001.
    Figure Legend Snippet: HMCES leads to a B cell intrinsic defect in CSR. A) Representative flow cytometry plots showing CSR to IgG1 and IgA in B cells isolated from Hmces +/+ and Hmces −/− mice and stimulated with anti-CD40, IL-4, TGF-β and IL-5 for 5 days. B) Bar graphs quantifying the kinetics of IgM to IgA switching in primary B cells from Hmces +/+ and Hmces −/− mice. C) Bar graphs quantifying the kinetics of IgM to IgG1 switching in primary B cells isolated from Hmces +/+ and Hmces −/− mice stimulated with anti-CD40, IL-4, TGF-β and IL-5. D) Bar graphs quantifying the kinetics of IgM to IgG1 switching in primary B cells isolated from Hmces +/+ and Hmces −/− mice and stimulated with anti-CD40 and IL-4. E) Bar graphs quantifying the kinetics of IgM to IgE switching in primary B cells isolated from Hmces +/+ and Hmces −/− mice and stimulated with anti-CD40 and IL-4. F) Bar graphs quantifying the kinetics of IgM to IgG3 switching in primary B cells isolated from Hmces +/+ and Hmces −/− mice and stimulated with LPS. Data are represented as mean ± SD. Each dot represents cells isolated from an individual mouse and lines are used to highlight samples analyzed in the same experiment. Statistical significance was calculated using two-way ANOVA. *p value ≤0.005, **p value ≤0.001.

    Techniques Used: Flow Cytometry, Isolation, Mouse Assay

    16) Product Images from "HMCES functions in the alternative end-joining pathway of the DNA DSB repair during class switch recombination in B cells."

    Article Title: HMCES functions in the alternative end-joining pathway of the DNA DSB repair during class switch recombination in B cells.

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2019.10.031

    HMCES deficiency results in defects in DNA DSB repair through the microhomology-mediated alternative end-joining pathway. A) Bar graphs quantifying the distribution of observed microhomologies in Sμ-Sα switch junctions of WT CH12, ΔHMCES CH12 clones H3 and H23, and ΔHMCES CH12 clone H3 reconstituted with FL HMCES protein (detected by PCR amplification and Sanger sequencing). Data are representative of two independent experiments. Statistical significance was computed using Welch’s T-test. B) Pie charts displaying distribution of Sμ-Sα switch region microhomologies of 0–3 or > 3 nucleotides in WT CH12, ΔHMCES CH12 clones H3 and H23, and ΔHMCES CH12 clone H3 reconstituted with FL HMCES. Cells were stimulated with anti-CD40, Interleukin-4 (IL-4) and Transforming growth factor-β (TGF-β) for 72 hours. C) Bar graphs quantifying the distribution of observed microhomologies in Sμ-Sγ1 switch junctions of Hmces +/+ and Hmces −/− primary B cells (detected by PCR amplification and Sanger sequencing) stimulated with anti-CD40 and IL-4 for 4 days. Data are representative of two independent experiments. Statistical significance was computed using Welch’s T-test. D) Pie charts displaying distribution of Sμ-Sγ1 switch junctional microhomologies of 0–3 or > 3 nucleotides in Hmces +/+ and Hmces −/− primary B cells stimulated with anti-CD40 and IL-4 for 4 days. E) Bar graphs showing the relative percentage of GFP-positive cells in ΔHMCES U2OS-EJ2-Alt-EJ reporter cells ( left ) or ΔHMCES U2OS-EJ5-cNHEJ reporter cells ( right ) compared with corresponding WT U2OS reporter cells. Data are represented as mean ± SD and are representative of at least three independent experiments. Statistical significance was calculated using student t-test. **p value ≤0.0001, *p value ≤0.0005.
    Figure Legend Snippet: HMCES deficiency results in defects in DNA DSB repair through the microhomology-mediated alternative end-joining pathway. A) Bar graphs quantifying the distribution of observed microhomologies in Sμ-Sα switch junctions of WT CH12, ΔHMCES CH12 clones H3 and H23, and ΔHMCES CH12 clone H3 reconstituted with FL HMCES protein (detected by PCR amplification and Sanger sequencing). Data are representative of two independent experiments. Statistical significance was computed using Welch’s T-test. B) Pie charts displaying distribution of Sμ-Sα switch region microhomologies of 0–3 or > 3 nucleotides in WT CH12, ΔHMCES CH12 clones H3 and H23, and ΔHMCES CH12 clone H3 reconstituted with FL HMCES. Cells were stimulated with anti-CD40, Interleukin-4 (IL-4) and Transforming growth factor-β (TGF-β) for 72 hours. C) Bar graphs quantifying the distribution of observed microhomologies in Sμ-Sγ1 switch junctions of Hmces +/+ and Hmces −/− primary B cells (detected by PCR amplification and Sanger sequencing) stimulated with anti-CD40 and IL-4 for 4 days. Data are representative of two independent experiments. Statistical significance was computed using Welch’s T-test. D) Pie charts displaying distribution of Sμ-Sγ1 switch junctional microhomologies of 0–3 or > 3 nucleotides in Hmces +/+ and Hmces −/− primary B cells stimulated with anti-CD40 and IL-4 for 4 days. E) Bar graphs showing the relative percentage of GFP-positive cells in ΔHMCES U2OS-EJ2-Alt-EJ reporter cells ( left ) or ΔHMCES U2OS-EJ5-cNHEJ reporter cells ( right ) compared with corresponding WT U2OS reporter cells. Data are represented as mean ± SD and are representative of at least three independent experiments. Statistical significance was calculated using student t-test. **p value ≤0.0001, *p value ≤0.0005.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Amplification, Sequencing

    HMCES deficiency leads to a defect in CSR in the CH12 B cell line. A) Representative flow cytometry plots of WT or ΔHMCES CH12 cells (clones H3 and H23). Cells were stimulated with anti-CD40, Interleukin-4 (IL-4) and Transforming growth factor-β (TGF-β) (CIT) and CSR from IgM to IgA was measured after 72 hours. B) Bar graph showing the relative percent CSR in ΔHMCES CH12 clones H3 and H23 compared with WT CH12 cells after 72 hours of CIT stimulation. C) Flow cytometry plots showing the frequency of CSR to IgA after 72 hours of CIT stimulation in WT and ΔHMCES CH12 cells transduced with either empty lentiviral vector or vector expressing full length (FL) HMCES protein. The ΔHMCES plot is representative of both H3 and H23 ΔHMCES CH12 cells. D) Bar graphs showing the relative percent CSR in ΔHMCES CH12 cells, normalized to WT CH12 cells transduced with empty vector in the same experiment. Data from H3 and H23 ΔHMCES CH12 cells are merged. E) Diagram indicating selected known features of human HMCES protein tested with the mutational analysis. F) Flow cytometry plots showing the frequency of CSR to IgA after 72 hours of CIT stimulation in WT and ΔHMCES CH12 cells transduced with either empty lentiviral vector or vectors expressing HMCES 1–270 (SRAPd), HMCES C2A or HMCES R212A variants generated on FL HMCES backbone. The ΔHMCES plot is representative of both H3 and H23 ΔHMCES CH12 cells. G) Bar graph showing the relative percent CSR of WT or ΔHMCES (both H3 and H23) CH12 cells lentivirally transduced with empty vector, HMCES 1–270 (SRAPd), HMCES C2A and HMCES R212A variant proteins generated on. CSR is normalized to WT CH12 cells transduced with empty vector in the same experiment. Data are representative of 4 or more independent experiments with each dot representing an independent experiment. Data are represented as mean ± SD. Statistical significance was calculated using student t-test. ***p value ≤0.000001, *p value ≤0.001 (panels B and D) or by two-way ANOVA **p value ≤0.0001 (panel G).
    Figure Legend Snippet: HMCES deficiency leads to a defect in CSR in the CH12 B cell line. A) Representative flow cytometry plots of WT or ΔHMCES CH12 cells (clones H3 and H23). Cells were stimulated with anti-CD40, Interleukin-4 (IL-4) and Transforming growth factor-β (TGF-β) (CIT) and CSR from IgM to IgA was measured after 72 hours. B) Bar graph showing the relative percent CSR in ΔHMCES CH12 clones H3 and H23 compared with WT CH12 cells after 72 hours of CIT stimulation. C) Flow cytometry plots showing the frequency of CSR to IgA after 72 hours of CIT stimulation in WT and ΔHMCES CH12 cells transduced with either empty lentiviral vector or vector expressing full length (FL) HMCES protein. The ΔHMCES plot is representative of both H3 and H23 ΔHMCES CH12 cells. D) Bar graphs showing the relative percent CSR in ΔHMCES CH12 cells, normalized to WT CH12 cells transduced with empty vector in the same experiment. Data from H3 and H23 ΔHMCES CH12 cells are merged. E) Diagram indicating selected known features of human HMCES protein tested with the mutational analysis. F) Flow cytometry plots showing the frequency of CSR to IgA after 72 hours of CIT stimulation in WT and ΔHMCES CH12 cells transduced with either empty lentiviral vector or vectors expressing HMCES 1–270 (SRAPd), HMCES C2A or HMCES R212A variants generated on FL HMCES backbone. The ΔHMCES plot is representative of both H3 and H23 ΔHMCES CH12 cells. G) Bar graph showing the relative percent CSR of WT or ΔHMCES (both H3 and H23) CH12 cells lentivirally transduced with empty vector, HMCES 1–270 (SRAPd), HMCES C2A and HMCES R212A variant proteins generated on. CSR is normalized to WT CH12 cells transduced with empty vector in the same experiment. Data are representative of 4 or more independent experiments with each dot representing an independent experiment. Data are represented as mean ± SD. Statistical significance was calculated using student t-test. ***p value ≤0.000001, *p value ≤0.001 (panels B and D) or by two-way ANOVA **p value ≤0.0001 (panel G).

    Techniques Used: Flow Cytometry, Clone Assay, Transduction, Plasmid Preparation, Expressing, Generated, Variant Assay

    Genetic disruption of c-NHEJ in HMCES-deficient CH12 cells results in a striking decrease in DNA DSB repair. A) Representative flow cytometry plots showing CSR to IgA in CH12 shNT, CH12 shKU70, ΔHMCES shNT and ΔHMCES shKU70 cells stimulated with anti-CD40, Interleukin-4 (IL-4) and Transforming growth factor-β (TGF-β) (CIT) for 72 hours. B) Bar graphs quantifying the relative percent of CSR to IgA in WT or ΔHMCES (H23) CH12 cells lentivirally transduced with a non-targeting shRNA (shNT) or shRNA against KU70 (shKU70). Percent switching is normalized to WT CH12 cells transduced with shNT in each experiment. C) Representative flow cytometry plots showing CSR to IgA in WT, ΔHMCES (H3 and H23), ΔLIGASE4 (CH12 L12) and, ΔHMCES and LIGASE4 double-deficient CH12 cells (H3 L15, H23 L2, H23 L18) after 72 hours stimulation with CIT. D) Bar graphs quantifying the relative percent CSR to IgA in WT, ΔHMCES (H3 and H23), ΔLIGASE4 (CH12 L12) and, ΔHMCES and LIGASE4 double-deficient CH12 cells (H3 L15, H23 L2, H23 L18). E) Representative flow cytometry plots showing switching to IgA in CH12 shNT, CH12 shCTIP-1, ΔHMCES shNT, ΔHMCES shCTIP-1 and ΔHMCES shCTIP-2 cells stimulated with CIT for 72 hours. F) Bar graphs quantifying CSR to IgA in WT or ΔHMCES (H23) CH12 cells lentivirally transduced with a non-targeting shRNA (shNT) or shRNAs against CTIP (shCTIP-1 and shCTIP-2). Percent switching is normalized to WT CH12 cells transduced with shNT in each experiment. Data in panels B and D are represented as mean ± SD and each dot represents an independent experiment. Statistical significance was calculated using student t-test. *p value ≤0.0005, **p value ≤0.00001.
    Figure Legend Snippet: Genetic disruption of c-NHEJ in HMCES-deficient CH12 cells results in a striking decrease in DNA DSB repair. A) Representative flow cytometry plots showing CSR to IgA in CH12 shNT, CH12 shKU70, ΔHMCES shNT and ΔHMCES shKU70 cells stimulated with anti-CD40, Interleukin-4 (IL-4) and Transforming growth factor-β (TGF-β) (CIT) for 72 hours. B) Bar graphs quantifying the relative percent of CSR to IgA in WT or ΔHMCES (H23) CH12 cells lentivirally transduced with a non-targeting shRNA (shNT) or shRNA against KU70 (shKU70). Percent switching is normalized to WT CH12 cells transduced with shNT in each experiment. C) Representative flow cytometry plots showing CSR to IgA in WT, ΔHMCES (H3 and H23), ΔLIGASE4 (CH12 L12) and, ΔHMCES and LIGASE4 double-deficient CH12 cells (H3 L15, H23 L2, H23 L18) after 72 hours stimulation with CIT. D) Bar graphs quantifying the relative percent CSR to IgA in WT, ΔHMCES (H3 and H23), ΔLIGASE4 (CH12 L12) and, ΔHMCES and LIGASE4 double-deficient CH12 cells (H3 L15, H23 L2, H23 L18). E) Representative flow cytometry plots showing switching to IgA in CH12 shNT, CH12 shCTIP-1, ΔHMCES shNT, ΔHMCES shCTIP-1 and ΔHMCES shCTIP-2 cells stimulated with CIT for 72 hours. F) Bar graphs quantifying CSR to IgA in WT or ΔHMCES (H23) CH12 cells lentivirally transduced with a non-targeting shRNA (shNT) or shRNAs against CTIP (shCTIP-1 and shCTIP-2). Percent switching is normalized to WT CH12 cells transduced with shNT in each experiment. Data in panels B and D are represented as mean ± SD and each dot represents an independent experiment. Statistical significance was calculated using student t-test. *p value ≤0.0005, **p value ≤0.00001.

    Techniques Used: Non-Homologous End Joining, Flow Cytometry, Transduction, shRNA

    HMCES leads to a B cell intrinsic defect in CSR. A) Representative flow cytometry plots showing CSR to IgG1 and IgA in B cells isolated from Hmces +/+ and Hmces −/− mice and stimulated with anti-CD40, IL-4, TGF-β and IL-5 for 5 days. B) Bar graphs quantifying the kinetics of IgM to IgA switching in primary B cells from Hmces +/+ and Hmces −/− mice. C) Bar graphs quantifying the kinetics of IgM to IgG1 switching in primary B cells isolated from Hmces +/+ and Hmces −/− mice stimulated with anti-CD40, IL-4, TGF-β and IL-5. D) Bar graphs quantifying the kinetics of IgM to IgG1 switching in primary B cells isolated from Hmces +/+ and Hmces −/− mice and stimulated with anti-CD40 and IL-4. E) Bar graphs quantifying the kinetics of IgM to IgE switching in primary B cells isolated from Hmces +/+ and Hmces −/− mice and stimulated with anti-CD40 and IL-4. F) Bar graphs quantifying the kinetics of IgM to IgG3 switching in primary B cells isolated from Hmces +/+ and Hmces −/− mice and stimulated with LPS. Data are represented as mean ± SD. Each dot represents cells isolated from an individual mouse and lines are used to highlight samples analyzed in the same experiment. Statistical significance was calculated using two-way ANOVA. *p value ≤0.005, **p value ≤0.001.
    Figure Legend Snippet: HMCES leads to a B cell intrinsic defect in CSR. A) Representative flow cytometry plots showing CSR to IgG1 and IgA in B cells isolated from Hmces +/+ and Hmces −/− mice and stimulated with anti-CD40, IL-4, TGF-β and IL-5 for 5 days. B) Bar graphs quantifying the kinetics of IgM to IgA switching in primary B cells from Hmces +/+ and Hmces −/− mice. C) Bar graphs quantifying the kinetics of IgM to IgG1 switching in primary B cells isolated from Hmces +/+ and Hmces −/− mice stimulated with anti-CD40, IL-4, TGF-β and IL-5. D) Bar graphs quantifying the kinetics of IgM to IgG1 switching in primary B cells isolated from Hmces +/+ and Hmces −/− mice and stimulated with anti-CD40 and IL-4. E) Bar graphs quantifying the kinetics of IgM to IgE switching in primary B cells isolated from Hmces +/+ and Hmces −/− mice and stimulated with anti-CD40 and IL-4. F) Bar graphs quantifying the kinetics of IgM to IgG3 switching in primary B cells isolated from Hmces +/+ and Hmces −/− mice and stimulated with LPS. Data are represented as mean ± SD. Each dot represents cells isolated from an individual mouse and lines are used to highlight samples analyzed in the same experiment. Statistical significance was calculated using two-way ANOVA. *p value ≤0.005, **p value ≤0.001.

    Techniques Used: Flow Cytometry, Isolation, Mouse Assay

    17) Product Images from "TRAF2 deficiency in B cells impairs CD40-induced isotype-switching that can be rescued by restoring NF-κB1 activation"

    Article Title: TRAF2 deficiency in B cells impairs CD40-induced isotype-switching that can be rescued by restoring NF-κB1 activation

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

    doi: 10.4049/jimmunol.1800337

    B cell-intrinsic TRAF2 but not TRAF3 deficiency impairs CD40-induced CSR ex vivo .
    Figure Legend Snippet: B cell-intrinsic TRAF2 but not TRAF3 deficiency impairs CD40-induced CSR ex vivo .

    Techniques Used: Ex Vivo

    PMA rescues the defective CD40-induced NF-κB1 complex activation and CSR in B-TRAF2-KO B cells.
    Figure Legend Snippet: PMA rescues the defective CD40-induced NF-κB1 complex activation and CSR in B-TRAF2-KO B cells.

    Techniques Used: Activation Assay

    Effects of B cell-intrinsic TRAF2 or TRAF3 deficiency on AID expression and GLT of Cγ1 or Cε induced by CD40.
    Figure Legend Snippet: Effects of B cell-intrinsic TRAF2 or TRAF3 deficiency on AID expression and GLT of Cγ1 or Cε induced by CD40.

    Techniques Used: Expressing

    TRAF2 is required for CD40 signaling to activate the NF-κB1 complex and induce isotype-switching.
    Figure Legend Snippet: TRAF2 is required for CD40 signaling to activate the NF-κB1 complex and induce isotype-switching.

    Techniques Used:

    Related Articles

    In Vitro:

    Article Title: Arhgap25 deficiency leads to decreased numbers of peripheral blood B cells and defective germinal center reactions
    Article Snippet: The number of migrated cells was evaluated using Precision Count Beads (424902, Biolegend, San Diego CA) by flow cytometry. .. In vitro B cell activationSplenic B cells of both genotypes were CD43-depleted using magnetic beads (130-049-801, Miltenyi Biotec, Bergisch Gladbach Germany) and taken in culture in the presence of 1μg/ml anti-CD40 antibody (102901, Biolegend, San Diego CA), 25ng/ml IL-4 (404-ML-025, R & D Systems, Minneapolis MN) and 25ng/ml IL-21(210–21, PeproTech, Rocky Hill NJ) for 4 days. .. Before culture, cells were labelled with 10μM Cell Proliferation Dye eFluor450 (65-0842-85, eBioscience, San Diego CA) according to manufacturer’s protocol.

    Article Title: Arhgap25 deficiency leads to decreased numbers of peripheral blood B cells and defective germinal center reactions
    Article Snippet: The number of migrated cells was evaluated using Precision Count Beads (424902, Biolegend, San Diego CA) by flow cytometry. .. In vitro B cell activationSplenic B cells of both genotypes were CD43-depleted using magnetic beads (130-049-801, Miltenyi Biotec, Bergisch Gladbach Germany) and taken in culture in the presence of 1μg/ml anti-CD40 antibody (102901, Biolegend, San Diego CA), 25ng/ml IL-4 (404-ML-025, R & D Systems, Minneapolis MN) and 25ng/ml IL-21(210-21, PeproTech, Rocky Hill NJ) for 4 days. .. Before culture, cells were labelled with 10μM Cell Proliferation Dye eFluor450 (65-0842-85, eBioscience, San Diego CA) according to manufacturer’s protocol.

    Article Title: Arhgap25 Deficiency Leads to Decreased Numbers of Peripheral Blood B Cells and Defective Germinal Center Reactions.
    Article Snippet: The number of migrated cells was evaluated using Precision Count Beads (424902; BioLegend) by flow cytometry. .. In vitro B cell activation Splenic B cells of both genotypes were CD43 depleted using magnetic beads (130-049-801; Miltenyi Biotec) and taken in culture in the presence of 1 mg/ml anti-CD40 Ab (102901; BioLegend), 25 ng/ml IL-4 (404-ML-025; R Systems, Minneapolis, MN), and 25 ng/ml IL-21 (210-21; PeproTech) for 4 d. Before culture, cells were labeled with 10 mM Cell Proliferation Dye eFluor450 (65-0842-85; eBioscience) according to manufacturer’s protocol. .. Cell division was monitored by flow cytometry, and the fraction of dividing plasmablasts was stained with CD138.

    Magnetic Beads:

    Article Title: Arhgap25 deficiency leads to decreased numbers of peripheral blood B cells and defective germinal center reactions
    Article Snippet: The number of migrated cells was evaluated using Precision Count Beads (424902, Biolegend, San Diego CA) by flow cytometry. .. In vitro B cell activationSplenic B cells of both genotypes were CD43-depleted using magnetic beads (130-049-801, Miltenyi Biotec, Bergisch Gladbach Germany) and taken in culture in the presence of 1μg/ml anti-CD40 antibody (102901, Biolegend, San Diego CA), 25ng/ml IL-4 (404-ML-025, R & D Systems, Minneapolis MN) and 25ng/ml IL-21(210–21, PeproTech, Rocky Hill NJ) for 4 days. .. Before culture, cells were labelled with 10μM Cell Proliferation Dye eFluor450 (65-0842-85, eBioscience, San Diego CA) according to manufacturer’s protocol.

    Article Title: Arhgap25 deficiency leads to decreased numbers of peripheral blood B cells and defective germinal center reactions
    Article Snippet: The number of migrated cells was evaluated using Precision Count Beads (424902, Biolegend, San Diego CA) by flow cytometry. .. In vitro B cell activationSplenic B cells of both genotypes were CD43-depleted using magnetic beads (130-049-801, Miltenyi Biotec, Bergisch Gladbach Germany) and taken in culture in the presence of 1μg/ml anti-CD40 antibody (102901, Biolegend, San Diego CA), 25ng/ml IL-4 (404-ML-025, R & D Systems, Minneapolis MN) and 25ng/ml IL-21(210-21, PeproTech, Rocky Hill NJ) for 4 days. .. Before culture, cells were labelled with 10μM Cell Proliferation Dye eFluor450 (65-0842-85, eBioscience, San Diego CA) according to manufacturer’s protocol.

    Article Title: Arhgap25 Deficiency Leads to Decreased Numbers of Peripheral Blood B Cells and Defective Germinal Center Reactions.
    Article Snippet: The number of migrated cells was evaluated using Precision Count Beads (424902; BioLegend) by flow cytometry. .. In vitro B cell activation Splenic B cells of both genotypes were CD43 depleted using magnetic beads (130-049-801; Miltenyi Biotec) and taken in culture in the presence of 1 mg/ml anti-CD40 Ab (102901; BioLegend), 25 ng/ml IL-4 (404-ML-025; R Systems, Minneapolis, MN), and 25 ng/ml IL-21 (210-21; PeproTech) for 4 d. Before culture, cells were labeled with 10 mM Cell Proliferation Dye eFluor450 (65-0842-85; eBioscience) according to manufacturer’s protocol. .. Cell division was monitored by flow cytometry, and the fraction of dividing plasmablasts was stained with CD138.

    Cell Culture:

    Article Title: Increased B Cell ADAM10 in Allergic Patients and Th2 Prone Mice
    Article Snippet: HDM Dermatophagoides pteronyssinus IgE was determined by ImmunoCAP (Phadia, 14-4107-01). .. Murine B cell culture and TNF ELISA Positively selected B220+ splenic B cells (Miltenyi Biotec) were grown for 1–3 days in cultures containing 1000 units IL-4 and 50 μg/mL LPS (E . coli 0111:B4, Sigma) or 1.25μg/mL purified anti-mouse CD40 (Biolegend) [ ]. .. Supernatants were analyzed for sTNF by ELISA (eBioscience).

    Enzyme-linked Immunosorbent Assay:

    Article Title: Increased B Cell ADAM10 in Allergic Patients and Th2 Prone Mice
    Article Snippet: HDM Dermatophagoides pteronyssinus IgE was determined by ImmunoCAP (Phadia, 14-4107-01). .. Murine B cell culture and TNF ELISA Positively selected B220+ splenic B cells (Miltenyi Biotec) were grown for 1–3 days in cultures containing 1000 units IL-4 and 50 μg/mL LPS (E . coli 0111:B4, Sigma) or 1.25μg/mL purified anti-mouse CD40 (Biolegend) [ ]. .. Supernatants were analyzed for sTNF by ELISA (eBioscience).

    Purification:

    Article Title: Increased B Cell ADAM10 in Allergic Patients and Th2 Prone Mice
    Article Snippet: HDM Dermatophagoides pteronyssinus IgE was determined by ImmunoCAP (Phadia, 14-4107-01). .. Murine B cell culture and TNF ELISA Positively selected B220+ splenic B cells (Miltenyi Biotec) were grown for 1–3 days in cultures containing 1000 units IL-4 and 50 μg/mL LPS (E . coli 0111:B4, Sigma) or 1.25μg/mL purified anti-mouse CD40 (Biolegend) [ ]. .. Supernatants were analyzed for sTNF by ELISA (eBioscience).

    Article Title: Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID
    Article Snippet: Assay of CSR in primary splenic B cells B cells were isolated from spleens of C57BL/6 mice and enriched through a negative selection in AUTOMACs with biotinylated anti-CD43 antibody (BD Pharmigen, Cat # 5532269) and streptavidin magnetic microbeads (Miltenyi Biotech, Cat # 130-048-102). .. Purified B cells were transduced for 24 hr in X-vivo medium (Lonza) containing 2 mM L-glutamine, 50 μM ß-mercaptoethanol, 5 ng/ml IL-4 (R & D Systems, cat# 404-ML-010) and 1 μg/mL anti-CD40 antibody (BioLegend, Cat# 102802) in 100 μl total volume in a round bottom 96-well plate, then transferred at 24 hr to supplemented RPMI (see above) containing 5 ng/ml IL-4 and 1 μg/ml anti-CD40 antibody. .. Cells were cultured for 4–5 days, stained with anti-IgG1 (FITC anti-mouse IgG1; BioLegend, Cat# 406605), and surface IgG1 quantified by flow-cytometry.

    Activation Assay:

    Article Title: Arhgap25 Deficiency Leads to Decreased Numbers of Peripheral Blood B Cells and Defective Germinal Center Reactions.
    Article Snippet: The number of migrated cells was evaluated using Precision Count Beads (424902; BioLegend) by flow cytometry. .. In vitro B cell activation Splenic B cells of both genotypes were CD43 depleted using magnetic beads (130-049-801; Miltenyi Biotec) and taken in culture in the presence of 1 mg/ml anti-CD40 Ab (102901; BioLegend), 25 ng/ml IL-4 (404-ML-025; R Systems, Minneapolis, MN), and 25 ng/ml IL-21 (210-21; PeproTech) for 4 d. Before culture, cells were labeled with 10 mM Cell Proliferation Dye eFluor450 (65-0842-85; eBioscience) according to manufacturer’s protocol. .. Cell division was monitored by flow cytometry, and the fraction of dividing plasmablasts was stained with CD138.

    Labeling:

    Article Title: Arhgap25 Deficiency Leads to Decreased Numbers of Peripheral Blood B Cells and Defective Germinal Center Reactions.
    Article Snippet: The number of migrated cells was evaluated using Precision Count Beads (424902; BioLegend) by flow cytometry. .. In vitro B cell activation Splenic B cells of both genotypes were CD43 depleted using magnetic beads (130-049-801; Miltenyi Biotec) and taken in culture in the presence of 1 mg/ml anti-CD40 Ab (102901; BioLegend), 25 ng/ml IL-4 (404-ML-025; R Systems, Minneapolis, MN), and 25 ng/ml IL-21 (210-21; PeproTech) for 4 d. Before culture, cells were labeled with 10 mM Cell Proliferation Dye eFluor450 (65-0842-85; eBioscience) according to manufacturer’s protocol. .. Cell division was monitored by flow cytometry, and the fraction of dividing plasmablasts was stained with CD138.

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    BioLegend anti cd40
    B cell-intrinsic TRAF2 but not TRAF3 deficiency impairs <t>CD40-induced</t> CSR ex vivo .
    Anti Cd40, supplied by BioLegend, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti cd40 - by Bioz Stars, 2021-05
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    BioLegend anti human cd40
    cGAMP up-regulates surface expression of activation markers on murine and human dendritic cells. ( A ) Murine bone marrow-derived DCs and ( B ) human PBMC-derived DCs were stimulated in vitro with c-di-AMP, cGAMP (both at ( A ) 5 µg/ml or ( B ) 60 µg/ml) or left untreated (mock) for 24 h. The DCs were decorated with fluorophore-conjugated antibodies against the DC activation markers <t>CD40,</t> CD54, CD80, CD83, CD86 or MHC class II (I-A b ) and analyzed by flow cytometry. Shown are the percentages of marker-positive CD11c + cells of the three independent experiments. Error bars are SEM of three independent experiments.
    Anti Human Cd40, supplied by BioLegend, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti human cd40/product/BioLegend
    Average 97 stars, based on 1 article reviews
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    anti human cd40 - by Bioz Stars, 2021-05
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    B cell-intrinsic TRAF2 but not TRAF3 deficiency impairs CD40-induced CSR ex vivo .

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

    Article Title: TRAF2 deficiency in B cells impairs CD40-induced isotype-switching that can be rescued by restoring NF-κB1 activation

    doi: 10.4049/jimmunol.1800337

    Figure Lengend Snippet: B cell-intrinsic TRAF2 but not TRAF3 deficiency impairs CD40-induced CSR ex vivo .

    Article Snippet: Purified B cells (0.5×106 /ml, 3 ml/well in 6-well plate) were stimulated in vitro with anti-CD40 (1μg/ml, Biolegend, clone HM40–3) plus IL-4 (10ng/ml, GenScript, Piscataway, NJ), or LPS (2μg/ml) plus IL-4 (10ng/ml) in 10% FBS RPMI lymphocyte medium for indicated days in a 5% CO2 incubator.

    Techniques: Ex Vivo

    PMA rescues the defective CD40-induced NF-κB1 complex activation and CSR in B-TRAF2-KO B cells.

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

    Article Title: TRAF2 deficiency in B cells impairs CD40-induced isotype-switching that can be rescued by restoring NF-κB1 activation

    doi: 10.4049/jimmunol.1800337

    Figure Lengend Snippet: PMA rescues the defective CD40-induced NF-κB1 complex activation and CSR in B-TRAF2-KO B cells.

    Article Snippet: Purified B cells (0.5×106 /ml, 3 ml/well in 6-well plate) were stimulated in vitro with anti-CD40 (1μg/ml, Biolegend, clone HM40–3) plus IL-4 (10ng/ml, GenScript, Piscataway, NJ), or LPS (2μg/ml) plus IL-4 (10ng/ml) in 10% FBS RPMI lymphocyte medium for indicated days in a 5% CO2 incubator.

    Techniques: Activation Assay

    Effects of B cell-intrinsic TRAF2 or TRAF3 deficiency on AID expression and GLT of Cγ1 or Cε induced by CD40.

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

    Article Title: TRAF2 deficiency in B cells impairs CD40-induced isotype-switching that can be rescued by restoring NF-κB1 activation

    doi: 10.4049/jimmunol.1800337

    Figure Lengend Snippet: Effects of B cell-intrinsic TRAF2 or TRAF3 deficiency on AID expression and GLT of Cγ1 or Cε induced by CD40.

    Article Snippet: Purified B cells (0.5×106 /ml, 3 ml/well in 6-well plate) were stimulated in vitro with anti-CD40 (1μg/ml, Biolegend, clone HM40–3) plus IL-4 (10ng/ml, GenScript, Piscataway, NJ), or LPS (2μg/ml) plus IL-4 (10ng/ml) in 10% FBS RPMI lymphocyte medium for indicated days in a 5% CO2 incubator.

    Techniques: Expressing

    TRAF2 is required for CD40 signaling to activate the NF-κB1 complex and induce isotype-switching.

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

    Article Title: TRAF2 deficiency in B cells impairs CD40-induced isotype-switching that can be rescued by restoring NF-κB1 activation

    doi: 10.4049/jimmunol.1800337

    Figure Lengend Snippet: TRAF2 is required for CD40 signaling to activate the NF-κB1 complex and induce isotype-switching.

    Article Snippet: Purified B cells (0.5×106 /ml, 3 ml/well in 6-well plate) were stimulated in vitro with anti-CD40 (1μg/ml, Biolegend, clone HM40–3) plus IL-4 (10ng/ml, GenScript, Piscataway, NJ), or LPS (2μg/ml) plus IL-4 (10ng/ml) in 10% FBS RPMI lymphocyte medium for indicated days in a 5% CO2 incubator.

    Techniques:

    PSGL-1 signaling inhibits in vitro activation of human peripheral B cells. (A) Relative frequencies of the different immunoglobulin (Ig)-expressing cell subsets in human total B cells (CD19 + ) and in the CD27 + and CD27 − B cell subpopulations. (B) Percentage of PSGL-1 + cells in the different Ig- expressing subsets of CD27 + and CD27 − B cells. (C) Relative frequencies of the different Ig- expressing cell subsets in the PSGL-1 + B cell subpopulation. (D) Percentage of surviving B cells after 72 h in culture: non-stimulated, activated with anti-IgM+anti-CD40 antibodies or activated with anti-IgM+anti-CD40+anti-PSGL-1 antibodies. (E) Relative frequency of CD27 + and CD27 − B cells in the total B cell population after 72 h in culture: non-stimulated, activated with anti-IgM+anti-CD40 antibodies or activated with anti-IgM+anti-CD40+anti-PSGL-1 antibodies. (F, G) Percentage of IL-10 + (F) and IgG + (G) cells in CD27 + and CD27 − B cells after 72 h in culture: non-stimulated, activated with anti-IgM+anti-CD40 antibodies or activated with anti-IgM+anti-CD40+anti-PSGL-1 antibodies. Bars represent the mean+standard deviation. *p

    Journal: Frontiers in Immunology

    Article Title: Relevance of PSGL-1 Expression in B Cell Development and Activation

    doi: 10.3389/fimmu.2020.588212

    Figure Lengend Snippet: PSGL-1 signaling inhibits in vitro activation of human peripheral B cells. (A) Relative frequencies of the different immunoglobulin (Ig)-expressing cell subsets in human total B cells (CD19 + ) and in the CD27 + and CD27 − B cell subpopulations. (B) Percentage of PSGL-1 + cells in the different Ig- expressing subsets of CD27 + and CD27 − B cells. (C) Relative frequencies of the different Ig- expressing cell subsets in the PSGL-1 + B cell subpopulation. (D) Percentage of surviving B cells after 72 h in culture: non-stimulated, activated with anti-IgM+anti-CD40 antibodies or activated with anti-IgM+anti-CD40+anti-PSGL-1 antibodies. (E) Relative frequency of CD27 + and CD27 − B cells in the total B cell population after 72 h in culture: non-stimulated, activated with anti-IgM+anti-CD40 antibodies or activated with anti-IgM+anti-CD40+anti-PSGL-1 antibodies. (F, G) Percentage of IL-10 + (F) and IgG + (G) cells in CD27 + and CD27 − B cells after 72 h in culture: non-stimulated, activated with anti-IgM+anti-CD40 antibodies or activated with anti-IgM+anti-CD40+anti-PSGL-1 antibodies. Bars represent the mean+standard deviation. *p

    Article Snippet: B cells were cultured in p96-dishes (200,000 cells/well) for 72 h in RPMI1640 10% FBS and activated with 5 μg/ml anti-IgM (BioLegend), 5 μg/ml anti-CD40 (BioLegend) and, if required, with 10 μg/ml anti-PSGL-1 (KPL1; BioLegend).

    Techniques: In Vitro, Activation Assay, Expressing, Standard Deviation

    TET proteins facilitate CSR. ( A ) Flowchart of in vivo experiment. f.p., footpad. ( B ) Top: Flow cytometric analysis of CD19 + GL7 + Fas + GC B cells at the draining popliteal lymph nodes from WT and Tet2/3 DKO mice after NP-OVA immunization as in (A). Bottom: Decreased IgG1-switched cells among GC B cells in Tet2/3 DKO (YFP + GC B–gated) compared with WT mice (GC B–gated). ( C to F ) Quantification of experiments shown in (B). Data shown are aggregated results from two independent experiments. Means and SEs are shown. WT, n = 11; DKO, n = 12. ( G ) Flowchart of in vitro IgG1 switching. Cells were labeled with Cell trace violet and activated for 4 days with LPS and IL-4. ( H and I ) Flow cytometry plots (H) and quantification (I) of IgG1-switched B cells in Tet2/3 DKO ( n = 4) and WT ( n = 4) mice. Representative of at least three independent experiments. ( J ) Circular γ1 transcript was quantified by qRT-PCR and normalized to Gapdh and then to the level of WT. Representative experiment of two independent experiments with three technical replicates. ( K ) Flowchart of in vitro IgA switching. Cells were activated for 5 days with anti-CD40, rmIL-4, rmIL-5, and rhTGFβ. ( L to N ) Flow cytometry plots (L) and quantification (M and N) of IgG1-switched (M) and IgA-switched cells (N). Representative of three independent experiments with three technical replicates. Statistical significance was calculated using unpaired two-tailed t test. * P

    Journal: Science immunology

    Article Title: TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer

    doi: 10.1126/sciimmunol.aau7523

    Figure Lengend Snippet: TET proteins facilitate CSR. ( A ) Flowchart of in vivo experiment. f.p., footpad. ( B ) Top: Flow cytometric analysis of CD19 + GL7 + Fas + GC B cells at the draining popliteal lymph nodes from WT and Tet2/3 DKO mice after NP-OVA immunization as in (A). Bottom: Decreased IgG1-switched cells among GC B cells in Tet2/3 DKO (YFP + GC B–gated) compared with WT mice (GC B–gated). ( C to F ) Quantification of experiments shown in (B). Data shown are aggregated results from two independent experiments. Means and SEs are shown. WT, n = 11; DKO, n = 12. ( G ) Flowchart of in vitro IgG1 switching. Cells were labeled with Cell trace violet and activated for 4 days with LPS and IL-4. ( H and I ) Flow cytometry plots (H) and quantification (I) of IgG1-switched B cells in Tet2/3 DKO ( n = 4) and WT ( n = 4) mice. Representative of at least three independent experiments. ( J ) Circular γ1 transcript was quantified by qRT-PCR and normalized to Gapdh and then to the level of WT. Representative experiment of two independent experiments with three technical replicates. ( K ) Flowchart of in vitro IgA switching. Cells were activated for 5 days with anti-CD40, rmIL-4, rmIL-5, and rhTGFβ. ( L to N ) Flow cytometry plots (L) and quantification (M and N) of IgG1-switched (M) and IgA-switched cells (N). Representative of three independent experiments with three technical replicates. Statistical significance was calculated using unpaired two-tailed t test. * P

    Article Snippet: For IgA switching, cells were activated with anti-CD40 (1 μg/ml, clone 1C10, BioLegend), rmIL-4 (10 ng/ml, Peprotech), rmIL-5 (10 ng/ml, Peprotech), and rhTGFβ1 (transforming growth factor β 1, 1 ng/ml).

    Techniques: In Vivo, Flow Cytometry, Mouse Assay, In Vitro, Labeling, Cytometry, Quantitative RT-PCR, Two Tailed Test

    cGAMP up-regulates surface expression of activation markers on murine and human dendritic cells. ( A ) Murine bone marrow-derived DCs and ( B ) human PBMC-derived DCs were stimulated in vitro with c-di-AMP, cGAMP (both at ( A ) 5 µg/ml or ( B ) 60 µg/ml) or left untreated (mock) for 24 h. The DCs were decorated with fluorophore-conjugated antibodies against the DC activation markers CD40, CD54, CD80, CD83, CD86 or MHC class II (I-A b ) and analyzed by flow cytometry. Shown are the percentages of marker-positive CD11c + cells of the three independent experiments. Error bars are SEM of three independent experiments.

    Journal: PLoS ONE

    Article Title: Cyclic GMP-AMP Displays Mucosal Adjuvant Activity in Mice

    doi: 10.1371/journal.pone.0110150

    Figure Lengend Snippet: cGAMP up-regulates surface expression of activation markers on murine and human dendritic cells. ( A ) Murine bone marrow-derived DCs and ( B ) human PBMC-derived DCs were stimulated in vitro with c-di-AMP, cGAMP (both at ( A ) 5 µg/ml or ( B ) 60 µg/ml) or left untreated (mock) for 24 h. The DCs were decorated with fluorophore-conjugated antibodies against the DC activation markers CD40, CD54, CD80, CD83, CD86 or MHC class II (I-A b ) and analyzed by flow cytometry. Shown are the percentages of marker-positive CD11c + cells of the three independent experiments. Error bars are SEM of three independent experiments.

    Article Snippet: Murine cells were decorated with anti-mouse CD80 (clone 16-10A1, APC-conjugated), CD86 (clone GL1, PE-conjugated), I-Ab (clone AF6-120.1, FITC-conjugated), CD11c (clone N418, PE-Cy7-conjugated); human cells with anti-human CD40 (clone 5C3, PE-conjugated), CD54 (clone HA58, APC-conjugated), CD80 (clone 2D10, APC-conjugated), CD83 (clone HB15e, PE-conjugated), CD86 (clone IT2.2, PE-Cy7-conjugated), CD11c (clone 3.9, Brilliant Violet 711-conjugated) (BioLegend, USA).

    Techniques: Expressing, Activation Assay, Derivative Assay, In Vitro, Flow Cytometry, Cytometry, Marker