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    LEGENDplex Human CD40L Capture Beads B9 13X
    Description:
    LEGENDplex Human CD40L Capture Beads B9 13X Apps Multiplex Size 100 tests 270 µl
    Catalog Number:
    740550
    Price:
    100
    Category:
    LEGENDplex
    Applications:
    Multiplex
    Conjugate:
    LEGENDplex
    Size:
    100 tests 270 µl
    Quantity:
    1
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    Structured Review

    BioLegend cd40
    GITR-expression modulates ILC1 functionality upon coculture with H1N1-infected BMDCs. BMDCs generated from wild-type mice using FLT-3 ligand stimulation were infected with the H1N1 PR8 strain (at a MOI of 1). In vitro -generated ILC1s were cocultured overnight with infected or uninfected BMDCs at a 1:1 ratio. The recombinant mouse GITR-Fc chimera protein was applied to the coculture overnight to manipulate GITR expression. Surface and cytokine staining were performed for flow cytometry analysis after 3 h of incubation in media with brefeldin and monensin. (A) GITR expression by ILC1s and BMDCs represented as MFI and representative histogram. (B) MFI of GITR expression by ILC1s with and without GITR-Fc treatment and representative histogram. MFI of (C) IFN-γ, (D) TNF-α, and (E) CD49a expression after GITR-Fc treated ILC1s cocultured with H1N1-infected BMDCs. (F) MFI of <t>CD40,</t> CD80, CD86, and MHC cl. II expression by infected BMDCs post-GITR-Fc treatment. Bars with scatter plots represent the mean ± SEM ( n = 3–4) and MFI data are representative from one out of two independent experiments. Asterisks denote significant values as calculated by nonparametric Mann–Whitney’s test; **** p ≤ 0.0001; *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05.
    LEGENDplex Human CD40L Capture Beads B9 13X Apps Multiplex Size 100 tests 270 µl
    https://www.bioz.com/result/cd40/product/BioLegend
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    cd40 - by Bioz Stars, 2021-05
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    Images

    1) Product Images from "Influenza-Activated ILC1s Contribute to Antiviral Immunity Partially Influenced by Differential GITR Expression"

    Article Title: Influenza-Activated ILC1s Contribute to Antiviral Immunity Partially Influenced by Differential GITR Expression

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00505

    GITR-expression modulates ILC1 functionality upon coculture with H1N1-infected BMDCs. BMDCs generated from wild-type mice using FLT-3 ligand stimulation were infected with the H1N1 PR8 strain (at a MOI of 1). In vitro -generated ILC1s were cocultured overnight with infected or uninfected BMDCs at a 1:1 ratio. The recombinant mouse GITR-Fc chimera protein was applied to the coculture overnight to manipulate GITR expression. Surface and cytokine staining were performed for flow cytometry analysis after 3 h of incubation in media with brefeldin and monensin. (A) GITR expression by ILC1s and BMDCs represented as MFI and representative histogram. (B) MFI of GITR expression by ILC1s with and without GITR-Fc treatment and representative histogram. MFI of (C) IFN-γ, (D) TNF-α, and (E) CD49a expression after GITR-Fc treated ILC1s cocultured with H1N1-infected BMDCs. (F) MFI of CD40, CD80, CD86, and MHC cl. II expression by infected BMDCs post-GITR-Fc treatment. Bars with scatter plots represent the mean ± SEM ( n = 3–4) and MFI data are representative from one out of two independent experiments. Asterisks denote significant values as calculated by nonparametric Mann–Whitney’s test; **** p ≤ 0.0001; *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05.
    Figure Legend Snippet: GITR-expression modulates ILC1 functionality upon coculture with H1N1-infected BMDCs. BMDCs generated from wild-type mice using FLT-3 ligand stimulation were infected with the H1N1 PR8 strain (at a MOI of 1). In vitro -generated ILC1s were cocultured overnight with infected or uninfected BMDCs at a 1:1 ratio. The recombinant mouse GITR-Fc chimera protein was applied to the coculture overnight to manipulate GITR expression. Surface and cytokine staining were performed for flow cytometry analysis after 3 h of incubation in media with brefeldin and monensin. (A) GITR expression by ILC1s and BMDCs represented as MFI and representative histogram. (B) MFI of GITR expression by ILC1s with and without GITR-Fc treatment and representative histogram. MFI of (C) IFN-γ, (D) TNF-α, and (E) CD49a expression after GITR-Fc treated ILC1s cocultured with H1N1-infected BMDCs. (F) MFI of CD40, CD80, CD86, and MHC cl. II expression by infected BMDCs post-GITR-Fc treatment. Bars with scatter plots represent the mean ± SEM ( n = 3–4) and MFI data are representative from one out of two independent experiments. Asterisks denote significant values as calculated by nonparametric Mann–Whitney’s test; **** p ≤ 0.0001; *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05.

    Techniques Used: Expressing, Infection, Generated, Mouse Assay, In Vitro, Recombinant, Staining, Flow Cytometry, Cytometry, Incubation, MANN-WHITNEY

    ILC1s are engaged in cross-talk with DCs during H1N1 infection in vitro . BMDCs generated from wild-type mice using the FLT-3 ligand stimulation were infected with the H1N1 PR8 strain (at a MOI of 1). In vitro -generated ILC1s cultured overnight with infected or uninfected BMDCs at a 1:1 ratio were stained for flow cytometry analysis after 3 h incubation in media with brefeldin and monensin. MFI of ILC1s expressing (A) IFN-γ, (B) TNF-α, (C) GITR, and (D) CD49a upon coculture with H1N1-infected or uninfected BMDCs. (E) Representative histograms for the expression of CD40, CD80, CD86, and MHC cl. II on infected BMDCs. (F) MFI of CD11c + BMDCs expressing CD40, CD80, CD86, and MHC cl. II after coculture with ILC1s. Bars with scatter plots represent the mean ± SEM ( n = 3–4) and MFI data are representative from one out of three independent experiments. Asterisks denote significant values as calculated by nonparametric Kruskal–Wallis test (Dunn’s posttest); **** p ≤ 0.0001; *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05.
    Figure Legend Snippet: ILC1s are engaged in cross-talk with DCs during H1N1 infection in vitro . BMDCs generated from wild-type mice using the FLT-3 ligand stimulation were infected with the H1N1 PR8 strain (at a MOI of 1). In vitro -generated ILC1s cultured overnight with infected or uninfected BMDCs at a 1:1 ratio were stained for flow cytometry analysis after 3 h incubation in media with brefeldin and monensin. MFI of ILC1s expressing (A) IFN-γ, (B) TNF-α, (C) GITR, and (D) CD49a upon coculture with H1N1-infected or uninfected BMDCs. (E) Representative histograms for the expression of CD40, CD80, CD86, and MHC cl. II on infected BMDCs. (F) MFI of CD11c + BMDCs expressing CD40, CD80, CD86, and MHC cl. II after coculture with ILC1s. Bars with scatter plots represent the mean ± SEM ( n = 3–4) and MFI data are representative from one out of three independent experiments. Asterisks denote significant values as calculated by nonparametric Kruskal–Wallis test (Dunn’s posttest); **** p ≤ 0.0001; *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05.

    Techniques Used: Infection, In Vitro, Generated, Mouse Assay, Cell Culture, Staining, Flow Cytometry, Cytometry, Incubation, Expressing

    2) Product Images from "MGL2+ Dermal Dendritic Cells Are Sufficient to Initiate Contact Hypersensitivity In Vivo"

    Article Title: MGL2+ Dermal Dendritic Cells Are Sufficient to Initiate Contact Hypersensitivity In Vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005619

    Expression of co-stimulatory molecules in MGL2 + DDC revealed by the flow cytometric analysis. (A) CD40 and CD86 staining on MGL2 + DDC in naive cutaneous LNs. MACS-purified CD11c + DCs were sub-divided into MGL2 + DDCs and MGL2 − DCs (left panel). Populations I-III indicate known cDC subsets in murine LNs. Note that MGL2 + DDCs were exclusively assigned to population III (CD11c hi CD40 hi DC). (B) Mean fluorescent intensity of CD40 (filled bars) and CD86 (open bars) staining of MGL2 + DDCs after FITC sensitization in the LN. * p
    Figure Legend Snippet: Expression of co-stimulatory molecules in MGL2 + DDC revealed by the flow cytometric analysis. (A) CD40 and CD86 staining on MGL2 + DDC in naive cutaneous LNs. MACS-purified CD11c + DCs were sub-divided into MGL2 + DDCs and MGL2 − DCs (left panel). Populations I-III indicate known cDC subsets in murine LNs. Note that MGL2 + DDCs were exclusively assigned to population III (CD11c hi CD40 hi DC). (B) Mean fluorescent intensity of CD40 (filled bars) and CD86 (open bars) staining of MGL2 + DDCs after FITC sensitization in the LN. * p

    Techniques Used: Expressing, Flow Cytometry, Staining, Magnetic Cell Separation, Purification

    3) Product Images from "Immunological Properties of Murine Parthenogenetic Stem Cell-Derived Cardiomyocytes and Engineered Heart Muscle"

    Article Title: Immunological Properties of Murine Parthenogenetic Stem Cell-Derived Cardiomyocytes and Engineered Heart Muscle

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00955

    Flow cytometric analysis of cell surface markers with immune functions on undirectedly differentiated pSC-derivatives (pSC-UD) and pSC-derived cardiomyocytes (pSC-CM) with and without IFN-γ-stimulation. Cell-fractions positive for major histocompatibility complex (MHC) class I and MHC class II molecules (A) , for programmed death ligand-1 (PDL-1) and PD-1 (B) , for co-stimulatory molecules CD40, CD80, and CD86 (C) and for CD1d (D) . * p
    Figure Legend Snippet: Flow cytometric analysis of cell surface markers with immune functions on undirectedly differentiated pSC-derivatives (pSC-UD) and pSC-derived cardiomyocytes (pSC-CM) with and without IFN-γ-stimulation. Cell-fractions positive for major histocompatibility complex (MHC) class I and MHC class II molecules (A) , for programmed death ligand-1 (PDL-1) and PD-1 (B) , for co-stimulatory molecules CD40, CD80, and CD86 (C) and for CD1d (D) . * p

    Techniques Used: Flow Cytometry, Derivative Assay

    Functional, morphological and immunological properties of parthenogenetic stem cell (pSC)-derived engineered heart muscle (EHM). (A) Contractile function of pSC-derived EHM (inset, bar graph: 500 µm) under increasing extracellular [Ca2+] ( n = 7). (B,C) Immunofluorescence stainings of whole mount EHMs. Immunolabeled structures are indicated in the respective panels. DNA (in blue) was labeled with Hoechst. Scale bars: 20 µm. (D–F) . FACS-analysis of cells derived from pSC-EHM for major histocompatibility complex (MHC) class I and MHC class II molecules (D) , for costimulatory molecules CD40, CD80, CD86 (E) , and for CD1d (F) with and without IFN-γ stimulation. * p
    Figure Legend Snippet: Functional, morphological and immunological properties of parthenogenetic stem cell (pSC)-derived engineered heart muscle (EHM). (A) Contractile function of pSC-derived EHM (inset, bar graph: 500 µm) under increasing extracellular [Ca2+] ( n = 7). (B,C) Immunofluorescence stainings of whole mount EHMs. Immunolabeled structures are indicated in the respective panels. DNA (in blue) was labeled with Hoechst. Scale bars: 20 µm. (D–F) . FACS-analysis of cells derived from pSC-EHM for major histocompatibility complex (MHC) class I and MHC class II molecules (D) , for costimulatory molecules CD40, CD80, CD86 (E) , and for CD1d (F) with and without IFN-γ stimulation. * p

    Techniques Used: Functional Assay, Derivative Assay, Immunofluorescence, Immunolabeling, Labeling, FACS

    4) Product Images from "Immunomodulatory Dual-Sized Microparticle System Conditions Human Antigen Presenting Cells Into a Tolerogenic Phenotype In Vitro and Inhibits Type 1 Diabetes-Specific Autoreactive T Cell Responses"

    Article Title: Immunomodulatory Dual-Sized Microparticle System Conditions Human Antigen Presenting Cells Into a Tolerogenic Phenotype In Vitro and Inhibits Type 1 Diabetes-Specific Autoreactive T Cell Responses

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.574447

    Dendritic cell modulation by dMP agents. ( A , upper panels) Representative gating schematic for live CD11c + DCs; (lower panels) representative histograms showing expression of activation markers HLA-DR, CD40, CD80, and CD86 under each culture condition (unstimulated (UN) in white, PLGA (gray) and VD3TGF (black) for gated DCs showing leftward shift in presence of dMP components. (B) Quantification of results depicted in (A) with calculated ratio of geometric mean fluorescence intensity (gMFI) in stimulated conditions (LPS) over unstimulated (UN) controls (n = 10) showing failure to upregulate activation markers in response to LPS with VD3TGF MPs in presence of GM-CSF. (C) IL-10 production in culture supernatants after (left) 72-h incubation with media (white circles), PLGA MPs (gray circles) or dMP (black circles), or 72-h incubation with treatments indicated as previous, with the addition of LPS (1 µg/ml) for the final 24 h of culture. dMP induced significantly increased IL-10 production over other treatments with or without inducing LPS stimulus. (D–G) Flow cytometry of replicate experiments for expression of negative regulators show increased intensity (gMFI) in presence of dMP for (D) PD-L1 (n = 14), (E) ILT-3 (n = 6), and (F) Galectin 9 with an apparent material associated increase in ILT-4 (G) . (*p
    Figure Legend Snippet: Dendritic cell modulation by dMP agents. ( A , upper panels) Representative gating schematic for live CD11c + DCs; (lower panels) representative histograms showing expression of activation markers HLA-DR, CD40, CD80, and CD86 under each culture condition (unstimulated (UN) in white, PLGA (gray) and VD3TGF (black) for gated DCs showing leftward shift in presence of dMP components. (B) Quantification of results depicted in (A) with calculated ratio of geometric mean fluorescence intensity (gMFI) in stimulated conditions (LPS) over unstimulated (UN) controls (n = 10) showing failure to upregulate activation markers in response to LPS with VD3TGF MPs in presence of GM-CSF. (C) IL-10 production in culture supernatants after (left) 72-h incubation with media (white circles), PLGA MPs (gray circles) or dMP (black circles), or 72-h incubation with treatments indicated as previous, with the addition of LPS (1 µg/ml) for the final 24 h of culture. dMP induced significantly increased IL-10 production over other treatments with or without inducing LPS stimulus. (D–G) Flow cytometry of replicate experiments for expression of negative regulators show increased intensity (gMFI) in presence of dMP for (D) PD-L1 (n = 14), (E) ILT-3 (n = 6), and (F) Galectin 9 with an apparent material associated increase in ILT-4 (G) . (*p

    Techniques Used: Expressing, Activation Assay, Fluorescence, Incubation, Flow Cytometry

    5) Product Images from "MGL2+ Dermal Dendritic Cells Are Sufficient to Initiate Contact Hypersensitivity In Vivo"

    Article Title: MGL2+ Dermal Dendritic Cells Are Sufficient to Initiate Contact Hypersensitivity In Vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005619

    Expression of co-stimulatory molecules in MGL2 + DDC revealed by the flow cytometric analysis. (A) CD40 and CD86 staining on MGL2 + DDC in naive cutaneous LNs. MACS-purified CD11c + DCs were sub-divided into MGL2 + DDCs and MGL2 − DCs (left panel). Populations I-III indicate known cDC subsets in murine LNs. Note that MGL2 + DDCs were exclusively assigned to population III (CD11c hi CD40 hi DC). (B) Mean fluorescent intensity of CD40 (filled bars) and CD86 (open bars) staining of MGL2 + DDCs after FITC sensitization in the LN. * p
    Figure Legend Snippet: Expression of co-stimulatory molecules in MGL2 + DDC revealed by the flow cytometric analysis. (A) CD40 and CD86 staining on MGL2 + DDC in naive cutaneous LNs. MACS-purified CD11c + DCs were sub-divided into MGL2 + DDCs and MGL2 − DCs (left panel). Populations I-III indicate known cDC subsets in murine LNs. Note that MGL2 + DDCs were exclusively assigned to population III (CD11c hi CD40 hi DC). (B) Mean fluorescent intensity of CD40 (filled bars) and CD86 (open bars) staining of MGL2 + DDCs after FITC sensitization in the LN. * p

    Techniques Used: Expressing, Flow Cytometry, Staining, Magnetic Cell Separation, Purification

    6) Product Images from "Influenza Virus Hemagglutinin Glycoproteins with Different N-Glycan Patterns Activate Dendritic Cells In Vitro"

    Article Title: Influenza Virus Hemagglutinin Glycoproteins with Different N-Glycan Patterns Activate Dendritic Cells In Vitro

    Journal: Journal of Virology

    doi: 10.1128/JVI.00452-16

    Expression of KIF-rH1HA-enhanced HLA-DR, CD40, CD83, and CD86 in human mDCs. Human mDC maturation status was evaluated at 48 h posttreatment with 10 μg/ml rH1HA, rH1HA (KIF), rH1HA (KIF+E), or PBS (negative control [NC]) or 100 ng/ml LPS (positive
    Figure Legend Snippet: Expression of KIF-rH1HA-enhanced HLA-DR, CD40, CD83, and CD86 in human mDCs. Human mDC maturation status was evaluated at 48 h posttreatment with 10 μg/ml rH1HA, rH1HA (KIF), rH1HA (KIF+E), or PBS (negative control [NC]) or 100 ng/ml LPS (positive

    Techniques Used: Expressing, Negative Control

    7) Product Images from "Annexin A1 on the Surface of Early Apoptotic Cells Suppresses CD8+ T Cell Immunity"

    Article Title: Annexin A1 on the Surface of Early Apoptotic Cells Suppresses CD8+ T Cell Immunity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0062449

    Annexin A1 inhibits DC activation in vivo . Apoptotic, mOVA-expressing Drosophila Schneider cells (aS2-mOVA) transfected with annexin A1 (AnxA1) or a control plasmid (Ctrl) were injected into mice. After 2 days, DC in lymph nodes of naïve (−) and S2-injected mice were analyzed for CD40 and CD86 expression by flow cytometry. Shown are quantifications of the MFI for CD40 and CD86 of total lymph node cells gated on CD11c + and MHC class II + cells *P
    Figure Legend Snippet: Annexin A1 inhibits DC activation in vivo . Apoptotic, mOVA-expressing Drosophila Schneider cells (aS2-mOVA) transfected with annexin A1 (AnxA1) or a control plasmid (Ctrl) were injected into mice. After 2 days, DC in lymph nodes of naïve (−) and S2-injected mice were analyzed for CD40 and CD86 expression by flow cytometry. Shown are quantifications of the MFI for CD40 and CD86 of total lymph node cells gated on CD11c + and MHC class II + cells *P

    Techniques Used: Activation Assay, In Vivo, Expressing, Transfection, Plasmid Preparation, Injection, Mouse Assay, Flow Cytometry, Cytometry

    8) Product Images from "Gut microbiota dependent anti-tumor immunity restricts melanoma growth in Rnf5−/− mice"

    Article Title: Gut microbiota dependent anti-tumor immunity restricts melanoma growth in Rnf5−/− mice

    Journal: Nature Communications

    doi: 10.1038/s41467-019-09525-y

    Enhanced antitumor immune responses in Rnf5 −/− mice. a Growth of YUMM1.5 ( Braf V600E :PTEN −/− :Cdkn2a −/− ) melanoma cells after subcutaneous injection of 10 6 cells into WT or Rnf5 −/− mice ( n = 5). b Quantification of tumor-infiltrating effector (CD44 hi ) CD4 + and CD8 + T cells and total CD45 + cells on day 24 after tumor injection ( n = 5). c Frequencies of tumor-infiltrating TNF-α-, IFN-γ-, and IL-2-producing CD4 + and CD8 + T cells on day 24 after tumor inoculation ( n = 5). d Quantification of tumor-infiltrating total DCs, pDCs, mDCs, and CD8α + DCs on day 24 after tumor inoculation ( n = 5). e Expression (mean fluorescence intensity, MFI) of MHC class II, CD40, CD80, and CD86 on tumor-infiltrating DCs (CD45 + CD11c + ) on day 24 after tumor inoculation ( n = 5). f Quantification of OT-I CD8 + T cells in tumor-draining lymph nodes (TdLN) and non-draining lymph nodes (ndLN) of CD45.1 + WT and Rnf5 −/− mice injected with B16-OVA melanoma cells (WT, n = 6; Rnf5 −/− , n = 5). g , h Growth of YUMM1.5 melanoma cells in mice injected i.p. with control IgG and anti-CD4 ( g ) or control IgG and anti-CD8 ( h ) depleting antibodies on days 0, 3, 6, 11, 16 ( n = 9). FACS analysis revealed > 90% depletion of blood CD4 + and CD8 + T cells on day 7 after tumor inoculation. i , Growth of YUMM1.5 melanoma cells in lethally irradiated bone marrow-reconstituted WT or Rnf5 −/− mice (arrow indicates bone marrow donor → recipient; n = 7). Data are representative of three independent experiments ( a – e ), two independent experiments ( f , i ) and one experiment ( g , h ) ≥5 mice per group. Graphs show the mean ± s.e.m. * P
    Figure Legend Snippet: Enhanced antitumor immune responses in Rnf5 −/− mice. a Growth of YUMM1.5 ( Braf V600E :PTEN −/− :Cdkn2a −/− ) melanoma cells after subcutaneous injection of 10 6 cells into WT or Rnf5 −/− mice ( n = 5). b Quantification of tumor-infiltrating effector (CD44 hi ) CD4 + and CD8 + T cells and total CD45 + cells on day 24 after tumor injection ( n = 5). c Frequencies of tumor-infiltrating TNF-α-, IFN-γ-, and IL-2-producing CD4 + and CD8 + T cells on day 24 after tumor inoculation ( n = 5). d Quantification of tumor-infiltrating total DCs, pDCs, mDCs, and CD8α + DCs on day 24 after tumor inoculation ( n = 5). e Expression (mean fluorescence intensity, MFI) of MHC class II, CD40, CD80, and CD86 on tumor-infiltrating DCs (CD45 + CD11c + ) on day 24 after tumor inoculation ( n = 5). f Quantification of OT-I CD8 + T cells in tumor-draining lymph nodes (TdLN) and non-draining lymph nodes (ndLN) of CD45.1 + WT and Rnf5 −/− mice injected with B16-OVA melanoma cells (WT, n = 6; Rnf5 −/− , n = 5). g , h Growth of YUMM1.5 melanoma cells in mice injected i.p. with control IgG and anti-CD4 ( g ) or control IgG and anti-CD8 ( h ) depleting antibodies on days 0, 3, 6, 11, 16 ( n = 9). FACS analysis revealed > 90% depletion of blood CD4 + and CD8 + T cells on day 7 after tumor inoculation. i , Growth of YUMM1.5 melanoma cells in lethally irradiated bone marrow-reconstituted WT or Rnf5 −/− mice (arrow indicates bone marrow donor → recipient; n = 7). Data are representative of three independent experiments ( a – e ), two independent experiments ( f , i ) and one experiment ( g , h ) ≥5 mice per group. Graphs show the mean ± s.e.m. * P

    Techniques Used: Mouse Assay, Injection, Expressing, Fluorescence, FACS, Irradiation

    9) Product Images from "Factors Produced by Macrophages Eliminating Apoptotic Cells Demonstrate Pro-Resolutive Properties and Terminate Ongoing Inflammation"

    Article Title: Factors Produced by Macrophages Eliminating Apoptotic Cells Demonstrate Pro-Resolutive Properties and Terminate Ongoing Inflammation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02586

    Antigen-presenting cells demonstrate reprogramming in vivo after SuperMApo treatment of CIA mice. (A,B) Costimulatory (CD80/CD86/CD40) and MHC-II molecule (IA/IE) mean florescence intensity (MFI) expressions, evaluated in spleen plasmacytoid DC (pDC), conventional DC (cDC) and macrophages (macro) 72 h and 10 days after SuperMApo or vehicle treatment of arthritic mice. Data from representative experiments showing cell marker expression from individual mouse (5 mice per group). Th17, Th1 and Treg CD4 + T cell polarizations by the same APC are also shown (bars represent mean ± s.e.m. of triplicates of APC isolated from each mouse and cultured with naïve CD4 + T cells). (C) Arthritis clinical score of mice receiving SuperMApo treatment or vehicle and clodronate-loaded (CL) or control liposomes (mean ± s.e.m., 5 mice per group). *** P
    Figure Legend Snippet: Antigen-presenting cells demonstrate reprogramming in vivo after SuperMApo treatment of CIA mice. (A,B) Costimulatory (CD80/CD86/CD40) and MHC-II molecule (IA/IE) mean florescence intensity (MFI) expressions, evaluated in spleen plasmacytoid DC (pDC), conventional DC (cDC) and macrophages (macro) 72 h and 10 days after SuperMApo or vehicle treatment of arthritic mice. Data from representative experiments showing cell marker expression from individual mouse (5 mice per group). Th17, Th1 and Treg CD4 + T cell polarizations by the same APC are also shown (bars represent mean ± s.e.m. of triplicates of APC isolated from each mouse and cultured with naïve CD4 + T cells). (C) Arthritis clinical score of mice receiving SuperMApo treatment or vehicle and clodronate-loaded (CL) or control liposomes (mean ± s.e.m., 5 mice per group). *** P

    Techniques Used: In Vivo, Mouse Assay, IA, Marker, Expressing, Isolation, Cell Culture

    10) Product Images from "Molecular and Functional Diversity of Distinct Subpopulations of the Stressed Insulin-Secreting Cell's Vesiculome"

    Article Title: Molecular and Functional Diversity of Distinct Subpopulations of the Stressed Insulin-Secreting Cell's Vesiculome

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.01814

    Modulation of co-stimulatory molecules in NOD bmDC by beta AB. NOD Shi mice derived bmDC were incubated for 18 h with AB, MV or sEV derived from 8 × 10 7 MIN6 cells. (A) Flow cytometric analysis of the expression of the CD40, MHC II, and CD86 activation markers. Histograms with dashed lines represent bmDC treated with AB derived from cytokine beta cells. Histograms with solid lines represent bmDC treated with AB from untreated beta cells. Gray histograms represent isotypic controls. Data from one representative out of 2–5 independent experiments are shown. (B) Scatter plots show the median fluorescence intensity (MFI) of co-stimulatory molecules expression in bmDC after AB treatment normalized to No EV controls (median with range). Tukey's range test * P
    Figure Legend Snippet: Modulation of co-stimulatory molecules in NOD bmDC by beta AB. NOD Shi mice derived bmDC were incubated for 18 h with AB, MV or sEV derived from 8 × 10 7 MIN6 cells. (A) Flow cytometric analysis of the expression of the CD40, MHC II, and CD86 activation markers. Histograms with dashed lines represent bmDC treated with AB derived from cytokine beta cells. Histograms with solid lines represent bmDC treated with AB from untreated beta cells. Gray histograms represent isotypic controls. Data from one representative out of 2–5 independent experiments are shown. (B) Scatter plots show the median fluorescence intensity (MFI) of co-stimulatory molecules expression in bmDC after AB treatment normalized to No EV controls (median with range). Tukey's range test * P

    Techniques Used: Mouse Assay, Derivative Assay, Incubation, Expressing, Activation Assay, Fluorescence

    11) Product Images from "Protein tyrosine phosphatase PTPN22 regulates LFA-1 dependent Th1 responses"

    Article Title: Protein tyrosine phosphatase PTPN22 regulates LFA-1 dependent Th1 responses

    Journal: Journal of Autoimmunity

    doi: 10.1016/j.jaut.2018.07.008

    Potentiated Th1 responses induced by Ptpn22 −/− BMDC are not explained by altered production of a secreted factor. (A) Day 6 WT and Ptpn22 −/− BMDC were pulsed for 24 h in the presence or absence of LPS. Cell free supernatants were assessed for IL-12p40, IL-12p70 and IL-6 by cytokine specific immunoassay. N = 11–15 independent experiments per group. NS = not significant determined by unpaired T-test. (B) Day 6 WT and Ptpn22 −/− LPS and OVA 323-339 pulsed BMDC were co-cultured with cell trace violet (CTV) labelled WT CD4 + OT-II T-cells for 24 h and cell free supernatants were assessed for IL-12p40 by immunoassay. N = 6–7 per group with each data point representing an individual WT or Ptpn22 −/− BMDC preparation, points are paired by the OT-II responding T-cells. (C) Day 6 WT and Ptpn22 −/− LPS and OVA 323-339 pulsed BMDC were co-cultured with CTV labelled WT CD4 + OT-II T-cells for 24 h. Cell surface expression of CD40L on CD3 + CD4 + T-cells was determined by flow cytometry. One representative flow plot of 3 independent experiments; T-cells co-cultured with WT BMDC (dashed line) or Ptpn22 −/− BMDC (solid line) (D) LPS pulsed WT and Ptpn22 −/− BMDC were stimulated for 24 h with plate bound anti-CD40 and cell free supernatants were assessed for IL-12p40 by immunoassay. Data are depicted as in IL-12p40 fold change from PBS control to anti-CD40. N = 4 independent experiments; bars represent mean ± s.d. (E) WT OT-II T-cells were cultured for 6 days on anti-CD3 and ant-CD28 in the presence of 50% conditioned media obtained from day 6 WT or Ptpn22 −/− BMDC:WT OT-II co-cultures. At day 6 of culture in the presence of conditioned media T-cells were restimulated for 6 h with PMA, ionomycin and monensin and the proportion of CD3 + CD4 + IFNγ + , IL-17 + , or TNFα + cells determined by intracellular flow cytometry. N = 3 independent experiments; bars represent mean ± s.d. NS = not significant, *p ≤ 0.05, **p ≤ 0.01 determined by unpaired T-test.
    Figure Legend Snippet: Potentiated Th1 responses induced by Ptpn22 −/− BMDC are not explained by altered production of a secreted factor. (A) Day 6 WT and Ptpn22 −/− BMDC were pulsed for 24 h in the presence or absence of LPS. Cell free supernatants were assessed for IL-12p40, IL-12p70 and IL-6 by cytokine specific immunoassay. N = 11–15 independent experiments per group. NS = not significant determined by unpaired T-test. (B) Day 6 WT and Ptpn22 −/− LPS and OVA 323-339 pulsed BMDC were co-cultured with cell trace violet (CTV) labelled WT CD4 + OT-II T-cells for 24 h and cell free supernatants were assessed for IL-12p40 by immunoassay. N = 6–7 per group with each data point representing an individual WT or Ptpn22 −/− BMDC preparation, points are paired by the OT-II responding T-cells. (C) Day 6 WT and Ptpn22 −/− LPS and OVA 323-339 pulsed BMDC were co-cultured with CTV labelled WT CD4 + OT-II T-cells for 24 h. Cell surface expression of CD40L on CD3 + CD4 + T-cells was determined by flow cytometry. One representative flow plot of 3 independent experiments; T-cells co-cultured with WT BMDC (dashed line) or Ptpn22 −/− BMDC (solid line) (D) LPS pulsed WT and Ptpn22 −/− BMDC were stimulated for 24 h with plate bound anti-CD40 and cell free supernatants were assessed for IL-12p40 by immunoassay. Data are depicted as in IL-12p40 fold change from PBS control to anti-CD40. N = 4 independent experiments; bars represent mean ± s.d. (E) WT OT-II T-cells were cultured for 6 days on anti-CD3 and ant-CD28 in the presence of 50% conditioned media obtained from day 6 WT or Ptpn22 −/− BMDC:WT OT-II co-cultures. At day 6 of culture in the presence of conditioned media T-cells were restimulated for 6 h with PMA, ionomycin and monensin and the proportion of CD3 + CD4 + IFNγ + , IL-17 + , or TNFα + cells determined by intracellular flow cytometry. N = 3 independent experiments; bars represent mean ± s.d. NS = not significant, *p ≤ 0.05, **p ≤ 0.01 determined by unpaired T-test.

    Techniques Used: Cell Culture, Expressing, Flow Cytometry, Cytometry

    Ptpn22 −/− BMDC promote Th1 responses in an LFA-1 dependent manner. (A) Day 6 WT and Ptpn22 −/− BMDC were pulsed for 24 h in the presence or absence of LPS. Cell surface expression of maturation markers was determined by flow cytometry. Median Fluorescent intensity (MFI) of CD86, CD40, MHCcII IA b , and CD54 (ICAM-1). N = 6–10 independent experiments; bar represents mean ± s.e.m, NS = not significant determined by unpaired T-test. (B) Day 6 WT and Ptpn22 −/− LPS and OVA 323-339 pulsed BMDC were co-cultured with cell trace violet (CTV) labelled WT CD4 + OT-II T-cells for 6 days in the presence or absence of anti-LFA1 or isotype control. At day 6 cells were T-cells were restimulated with a fresh preparation of WT or Ptpn22 −/− BMDC for 48 h and then restimulated for 6 h with PMA, ionomycin and monensin and the proportion of CD3 + CD4 + IFNγ + or TNFα + cells was determined by intracellular flow cytometry. N = 4 independent experiments; bar represents mean ± s.d, NS = not significant, *p ≤ 0.05, determined by unpaired T-test.
    Figure Legend Snippet: Ptpn22 −/− BMDC promote Th1 responses in an LFA-1 dependent manner. (A) Day 6 WT and Ptpn22 −/− BMDC were pulsed for 24 h in the presence or absence of LPS. Cell surface expression of maturation markers was determined by flow cytometry. Median Fluorescent intensity (MFI) of CD86, CD40, MHCcII IA b , and CD54 (ICAM-1). N = 6–10 independent experiments; bar represents mean ± s.e.m, NS = not significant determined by unpaired T-test. (B) Day 6 WT and Ptpn22 −/− LPS and OVA 323-339 pulsed BMDC were co-cultured with cell trace violet (CTV) labelled WT CD4 + OT-II T-cells for 6 days in the presence or absence of anti-LFA1 or isotype control. At day 6 cells were T-cells were restimulated with a fresh preparation of WT or Ptpn22 −/− BMDC for 48 h and then restimulated for 6 h with PMA, ionomycin and monensin and the proportion of CD3 + CD4 + IFNγ + or TNFα + cells was determined by intracellular flow cytometry. N = 4 independent experiments; bar represents mean ± s.d, NS = not significant, *p ≤ 0.05, determined by unpaired T-test.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, IA, Cell Culture

    12) Product Images from "Soluble Antigen Arrays Displaying Mimotopes Direct the Response of Diabetogenic T cells"

    Article Title: Soluble Antigen Arrays Displaying Mimotopes Direct the Response of Diabetogenic T cells

    Journal: ACS chemical biology

    doi: 10.1021/acschembio.9b00090

    Uptake efficiency of f cSAgA p79 (1 μM) by different APCs at 12h ( A ) and 24h ( B ) time points and upregulation of costimulatory molecules CD40 ( C ) and CD86 ( D ) at 24h in splenocytes from BDC2.5 as compared to NOD or untreated control splenocytes. Control cells from NOD mice are not shown (comparable to BDC2.5 control). The values are given as mean ± SD (n=3 technical replicates) and the data are representative of two experiments performed with f cSAgA p79 , and experiments performed with FITC-conjugated SAgA p79 . MMN: monocytes, macrophages, neutrophils.
    Figure Legend Snippet: Uptake efficiency of f cSAgA p79 (1 μM) by different APCs at 12h ( A ) and 24h ( B ) time points and upregulation of costimulatory molecules CD40 ( C ) and CD86 ( D ) at 24h in splenocytes from BDC2.5 as compared to NOD or untreated control splenocytes. Control cells from NOD mice are not shown (comparable to BDC2.5 control). The values are given as mean ± SD (n=3 technical replicates) and the data are representative of two experiments performed with f cSAgA p79 , and experiments performed with FITC-conjugated SAgA p79 . MMN: monocytes, macrophages, neutrophils.

    Techniques Used: Mouse Assay

    13) Product Images from "Systemic viral spreading and defective host responses are associated with fatal Lassa fever in macaques"

    Article Title: Systemic viral spreading and defective host responses are associated with fatal Lassa fever in macaques

    Journal: Communications Biology

    doi: 10.1038/s42003-020-01543-7

    Analysis of circulating innate immune cells after LASV challenge. a The number of leukocytes, granulocytes, mDC (HLA-DR + CD14 − CD1c + and HLA-DR + CD14 − CD11c + ), and monocytes (HLA-DR + CD14 + ) in the blood is presented according to the time after LASV infection. The percentage of CD10 − cells among granulocytes is also presented. The percentage of monocytes expressing CD80, CD86, or CD40 is shown. Results show the mean ± standard error of the mean (SEM) for each group: controls ( n = 3), LASV-Josiah ( n = 6, except for day 14 where n = 2) and AV-infected animals ( n = 4) were analyzed for leukocyte, granulocyte, CD10 − , and monocyte numbers. For mDC, CD80, CD86, and CD40 analysis, six AV-infected animals were analyzed from day 0 to 6 and three of them from day 8 to 11. b The number of circulating NK cells (CD8 + CD3 − CD20 − cells) is presented, as well as the percentage of KI67 + , CD107a + , NKp80 + , and NKG2D + cells among NK cells ( n = 3 for controls, n = 6 for LASV-Josiah, and n = 4 for AV-infected animals). Statistical analyses were performed and are presented as in Fig. 1 . Individual values can be found in Supplementary data 1 for a and b. c The proportion of NK cells (CD8 + CD3 − CD20 − ) expressing KI67, (granzyme B) GrzB, CD107a, CXCR3, and NKp80 was quantified in spleen (S, upper graphs) and MLN (L, lower graphs) of controls ( n = 3), AV- ( n = 3), and Josiah-infected ( n = 3) animals, as well as the percentage of CD16 + CD56 − cells among NK cells. Individual values and mean ± SEM are expressed for each group. Statistical analyses were performed and are presented as in Fig. 1 .
    Figure Legend Snippet: Analysis of circulating innate immune cells after LASV challenge. a The number of leukocytes, granulocytes, mDC (HLA-DR + CD14 − CD1c + and HLA-DR + CD14 − CD11c + ), and monocytes (HLA-DR + CD14 + ) in the blood is presented according to the time after LASV infection. The percentage of CD10 − cells among granulocytes is also presented. The percentage of monocytes expressing CD80, CD86, or CD40 is shown. Results show the mean ± standard error of the mean (SEM) for each group: controls ( n = 3), LASV-Josiah ( n = 6, except for day 14 where n = 2) and AV-infected animals ( n = 4) were analyzed for leukocyte, granulocyte, CD10 − , and monocyte numbers. For mDC, CD80, CD86, and CD40 analysis, six AV-infected animals were analyzed from day 0 to 6 and three of them from day 8 to 11. b The number of circulating NK cells (CD8 + CD3 − CD20 − cells) is presented, as well as the percentage of KI67 + , CD107a + , NKp80 + , and NKG2D + cells among NK cells ( n = 3 for controls, n = 6 for LASV-Josiah, and n = 4 for AV-infected animals). Statistical analyses were performed and are presented as in Fig. 1 . Individual values can be found in Supplementary data 1 for a and b. c The proportion of NK cells (CD8 + CD3 − CD20 − ) expressing KI67, (granzyme B) GrzB, CD107a, CXCR3, and NKp80 was quantified in spleen (S, upper graphs) and MLN (L, lower graphs) of controls ( n = 3), AV- ( n = 3), and Josiah-infected ( n = 3) animals, as well as the percentage of CD16 + CD56 − cells among NK cells. Individual values and mean ± SEM are expressed for each group. Statistical analyses were performed and are presented as in Fig. 1 .

    Techniques Used: Infection, Expressing

    14) Product Images from "MiR-125b potentiates macrophage activation 1"

    Article Title: MiR-125b potentiates macrophage activation 1

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

    doi: 10.4049/jimmunol.1102001

    MiR-125b increases macrophage response to IFNγ. A) Surface expression of MHCII, CD40, CD86, and CD80 in response to media alone or IFNγ. A representative flow cytometric plot of the IFNγ treated samples is shown for each factor.
    Figure Legend Snippet: MiR-125b increases macrophage response to IFNγ. A) Surface expression of MHCII, CD40, CD86, and CD80 in response to media alone or IFNγ. A representative flow cytometric plot of the IFNγ treated samples is shown for each factor.

    Techniques Used: Expressing, Flow Cytometry

    15) Product Images from "Autophagy is involved in regulating the immune response of dendritic cells to influenza A (H1N1) pdm09 infection"

    Article Title: Autophagy is involved in regulating the immune response of dendritic cells to influenza A (H1N1) pdm09 infection

    Journal: Immunology

    doi: 10.1111/imm.12587

    Beclin‐1 +/− bone‐marrow‐derived dendritic cells (BMDCs) are deficient in maturation and the production of innate and adaptive cytokines upon H1N1virus infection. (a, b) BMDCs from wild‐type (WT) or Beclin‐1 +/− mice were infected with H1N1 virus at a multiplicity of infection (MOI) of 2 for 2 hr. The LC3B‐positive puncta in BMDCs were detected by fluorescence staining (a), and the LC3B‐I/II and p62 proteins were detected by Western blot (b). (c) BMDCs from WT or Beclin‐1 +/− mice were infected with H1N1 virus at an MOI of 2 for 24 hr. The expression of MHC‐II, CD80, CD86 and CD40 was detected by flow cytometry. (d) Statistical analysis of the mean fluorescent intensity (MFI) of (c). (e) BMDCs from WT or Beclin‐1 +/− mice were infected with H1N1 virus at an MOI of 2 for 24 hr. The secretion of interleukin‐6 (IL‐6), tumour necrosis factor‐ α (TNF‐ α ), interferon‐ β (IFN‐ β ), IL‐12p70 and IFN‐ γ was measured by ELISA. (f) The H1N1 virus yield by BMDCs from WT or Beclin‐1 +/− mice at the indicated time‐points after H1N1 virus infection were determined by TCID 50 assay. (g) BMDCs from WT or Beclin‐1 +/− mice were cultured in Hanks' balanced salt solution medium for 2 hr then were infected by H1N1 virus at an MOI of 2 for 24 hr. The secretion of IL‐6, TNF‐ α , IFN‐ β , IL‐12p70 and IFN‐ γ was examined by ELISA. The data are representative of three independent experiments and are expressed as the means ± SEM. Statistical significance was determined using an unpaired Student's t ‐test for (d) and (e), and (g) was analysed by one‐way analysis of variance and Q test. * P
    Figure Legend Snippet: Beclin‐1 +/− bone‐marrow‐derived dendritic cells (BMDCs) are deficient in maturation and the production of innate and adaptive cytokines upon H1N1virus infection. (a, b) BMDCs from wild‐type (WT) or Beclin‐1 +/− mice were infected with H1N1 virus at a multiplicity of infection (MOI) of 2 for 2 hr. The LC3B‐positive puncta in BMDCs were detected by fluorescence staining (a), and the LC3B‐I/II and p62 proteins were detected by Western blot (b). (c) BMDCs from WT or Beclin‐1 +/− mice were infected with H1N1 virus at an MOI of 2 for 24 hr. The expression of MHC‐II, CD80, CD86 and CD40 was detected by flow cytometry. (d) Statistical analysis of the mean fluorescent intensity (MFI) of (c). (e) BMDCs from WT or Beclin‐1 +/− mice were infected with H1N1 virus at an MOI of 2 for 24 hr. The secretion of interleukin‐6 (IL‐6), tumour necrosis factor‐ α (TNF‐ α ), interferon‐ β (IFN‐ β ), IL‐12p70 and IFN‐ γ was measured by ELISA. (f) The H1N1 virus yield by BMDCs from WT or Beclin‐1 +/− mice at the indicated time‐points after H1N1 virus infection were determined by TCID 50 assay. (g) BMDCs from WT or Beclin‐1 +/− mice were cultured in Hanks' balanced salt solution medium for 2 hr then were infected by H1N1 virus at an MOI of 2 for 24 hr. The secretion of IL‐6, TNF‐ α , IFN‐ β , IL‐12p70 and IFN‐ γ was examined by ELISA. The data are representative of three independent experiments and are expressed as the means ± SEM. Statistical significance was determined using an unpaired Student's t ‐test for (d) and (e), and (g) was analysed by one‐way analysis of variance and Q test. * P

    Techniques Used: Derivative Assay, Infection, Mouse Assay, Fluorescence, Staining, Western Blot, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture

    16) Product Images from "Induction of NKG2D ligand expression on tumor cells by CD8+ T cell engagement-mediated activation of nuclear factor-kappa B and p300/CBP-associated factor"

    Article Title: Induction of NKG2D ligand expression on tumor cells by CD8+ T cell engagement-mediated activation of nuclear factor-kappa B and p300/CBP-associated factor

    Journal: Oncogene

    doi: 10.1038/s41388-019-0960-x

    Inflammatory cytokines are crucial for CD8 + T cell–mediated Rae-1 upregulation on tumor cells. (A-D) CT26 (A-B) and K7M3 (C-D) cells were co-cultured with stimulated CD8 + T cells or, for comparison, treated with TNFSRF CD137 and CD40 ligand recombinant proteins (Ag) in the presence or absence of CD8 + T cell–conditioned medium (CM). NF-κB signaling and Rae-1 expression were assessed via immunoblotting (A, C) and flow cytometry (B, D), respectively. MFI, mean fluorescence intensity. Bar graphs show means ± SEM. Values under each band indicate the density of protein expression. Results are representative of three repeated experiments. (E) LLC tumor cells were labeled with CFSE and cultured in medium conditioned with wildtype CD8 + T cells, IFNγ-knockout (KO) CD8 + T cells, or TNFαKO CD8 + T cells in the presence of recombinant CD40L (0.5 μg/mL) and CD137 (10 μg/mL) proteins. LLC cells alone were used as a negative control, and LLCs co-incubated with wildtype CD8 + T cells were used as a positive control. Rae-1 expression was determined by using flow cytometry. (F, G) LLC cells were labeled with CFSE and cultured in IFNγKO CD8 + T cell CM (F) or TNFαKO CD8 + T cell CM (G) in the presence or absence of IFNγ (10 ng/mL) (F) or TNFα (10 ng/mL) (G), respectively. LLC cells alone were used as a negative control, and LLCs co-incubated with wildtype CD8 + T cells were used as a positive control. Rae-1 expression was determined by using flow cytometry. iso ctrl: isotype control. Immunoblots of PCAF after each treatment was at the bottom panel. Bar graphs show means ± SEM. Results are representative of three repeated experiments. * P
    Figure Legend Snippet: Inflammatory cytokines are crucial for CD8 + T cell–mediated Rae-1 upregulation on tumor cells. (A-D) CT26 (A-B) and K7M3 (C-D) cells were co-cultured with stimulated CD8 + T cells or, for comparison, treated with TNFSRF CD137 and CD40 ligand recombinant proteins (Ag) in the presence or absence of CD8 + T cell–conditioned medium (CM). NF-κB signaling and Rae-1 expression were assessed via immunoblotting (A, C) and flow cytometry (B, D), respectively. MFI, mean fluorescence intensity. Bar graphs show means ± SEM. Values under each band indicate the density of protein expression. Results are representative of three repeated experiments. (E) LLC tumor cells were labeled with CFSE and cultured in medium conditioned with wildtype CD8 + T cells, IFNγ-knockout (KO) CD8 + T cells, or TNFαKO CD8 + T cells in the presence of recombinant CD40L (0.5 μg/mL) and CD137 (10 μg/mL) proteins. LLC cells alone were used as a negative control, and LLCs co-incubated with wildtype CD8 + T cells were used as a positive control. Rae-1 expression was determined by using flow cytometry. (F, G) LLC cells were labeled with CFSE and cultured in IFNγKO CD8 + T cell CM (F) or TNFαKO CD8 + T cell CM (G) in the presence or absence of IFNγ (10 ng/mL) (F) or TNFα (10 ng/mL) (G), respectively. LLC cells alone were used as a negative control, and LLCs co-incubated with wildtype CD8 + T cells were used as a positive control. Rae-1 expression was determined by using flow cytometry. iso ctrl: isotype control. Immunoblots of PCAF after each treatment was at the bottom panel. Bar graphs show means ± SEM. Results are representative of three repeated experiments. * P

    Techniques Used: Cell Culture, Recombinant, Expressing, Flow Cytometry, Fluorescence, Labeling, Knock-Out, Negative Control, Incubation, Positive Control, Western Blot

    Illustration of the mechanism of CD8 + T cell engagement-induced Rae-1 expression on tumor cells. Rae-1 upregulation was through CD137L/CD40 downstream NF-κB signaling and PCAF activation on tumor cells.
    Figure Legend Snippet: Illustration of the mechanism of CD8 + T cell engagement-induced Rae-1 expression on tumor cells. Rae-1 upregulation was through CD137L/CD40 downstream NF-κB signaling and PCAF activation on tumor cells.

    Techniques Used: Expressing, Activation Assay

    CD8 + T cells engage with tumor cells through TNFRSF to boost NKG2D ligand expression. (A, B) Murine CT26 colon carcinoma (A) and K7M3 osteosarcoma (B) cells were stained with CFSE and co-incubated with sham, activated CD8 + T cells (CD3/CD8 Dynabeads, IL-2 [50 U/mL], and IL-12 [10 ng/mL]) at a 1:1 tumor to T cell ratio, or CD8 + T cell–conditioned medium (CM) for 24 h. Rae-1 expression on tumor cells was determined by flow cytometry. Bar graphs show means ± standard error of the mean (SEM). MFI, mean fluorescence intensity. (C, D) CT26 (C) and K7M3 (D) cells were stained with CFSE and cultured in regular medium (RM) or CD8 + T cell CM. Expression of CD80, CD86, ICOSL, CD40, CD137L, OX40, and CD70 was determined by flow cytometry. (E, F) Activated CD8 + T cells were pretreated with control IgG, anti-CD137 (5 ng/mL), anti-CD40L (5 ng/mL), or anti-CD137 plus anti-CD40L for 3 h. CT26 (E) and K7M3 (F) cells were stained with CFSE and co-incubated with sham or pretreated CD8 + T cells for 24 h. Rae-1 expression on tumor cells was determined by flow cytometry. iso ctrl: isotype control. Bar graphs show means ± SEM. Results are representative of three repeated experiments. * P
    Figure Legend Snippet: CD8 + T cells engage with tumor cells through TNFRSF to boost NKG2D ligand expression. (A, B) Murine CT26 colon carcinoma (A) and K7M3 osteosarcoma (B) cells were stained with CFSE and co-incubated with sham, activated CD8 + T cells (CD3/CD8 Dynabeads, IL-2 [50 U/mL], and IL-12 [10 ng/mL]) at a 1:1 tumor to T cell ratio, or CD8 + T cell–conditioned medium (CM) for 24 h. Rae-1 expression on tumor cells was determined by flow cytometry. Bar graphs show means ± standard error of the mean (SEM). MFI, mean fluorescence intensity. (C, D) CT26 (C) and K7M3 (D) cells were stained with CFSE and cultured in regular medium (RM) or CD8 + T cell CM. Expression of CD80, CD86, ICOSL, CD40, CD137L, OX40, and CD70 was determined by flow cytometry. (E, F) Activated CD8 + T cells were pretreated with control IgG, anti-CD137 (5 ng/mL), anti-CD40L (5 ng/mL), or anti-CD137 plus anti-CD40L for 3 h. CT26 (E) and K7M3 (F) cells were stained with CFSE and co-incubated with sham or pretreated CD8 + T cells for 24 h. Rae-1 expression on tumor cells was determined by flow cytometry. iso ctrl: isotype control. Bar graphs show means ± SEM. Results are representative of three repeated experiments. * P

    Techniques Used: Expressing, Staining, Incubation, Flow Cytometry, Fluorescence, Cell Culture

    17) Product Images from "Glycyrrhizin Attenuates Salmonella enterica Serovar Typhimurium Infection: New Insights Into Its Protective Mechanism"

    Article Title: Glycyrrhizin Attenuates Salmonella enterica Serovar Typhimurium Infection: New Insights Into Its Protective Mechanism

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02321

    GL improves surface marker expression in BMDCs. BMDCs were treated with PBS (blank control), GL (200 μg/ml), or LPS (50 ng/ml; positive control) for 48 h. Cells were stained with antibodies against CD40 (A) , CD80 (B) , CD83 (C) , CD86 (D) , and MHC-II (E) , and the fluorescence signals were determined immediately using a FACScan flow cytometer. At least 10,000 events were collected from the cell gate. The data represent three independent experiments.
    Figure Legend Snippet: GL improves surface marker expression in BMDCs. BMDCs were treated with PBS (blank control), GL (200 μg/ml), or LPS (50 ng/ml; positive control) for 48 h. Cells were stained with antibodies against CD40 (A) , CD80 (B) , CD83 (C) , CD86 (D) , and MHC-II (E) , and the fluorescence signals were determined immediately using a FACScan flow cytometer. At least 10,000 events were collected from the cell gate. The data represent three independent experiments.

    Techniques Used: Marker, Expressing, Positive Control, Staining, Fluorescence, Flow Cytometry, Cytometry

    18) Product Images from "Lipid-laden partially-activated plasmacytoid and CD4−CD8α+ dendritic cells accumulate in tissues in elderly mice"

    Article Title: Lipid-laden partially-activated plasmacytoid and CD4−CD8α+ dendritic cells accumulate in tissues in elderly mice

    Journal: Immunity & Ageing : I & A

    doi: 10.1186/1742-4933-11-11

    pDC and CD4 - CD8α + DC activation status is influenced by age. The activation status of pDCs and CD4 - CD8α + DCs was evaluated by co-expression of CD40 (A and D) , MHC-I (B and E) and MHC-II (C and F) . Representative data from 1 of 2 experiments is shown as individual mean fluorescent intensities (MFIs) and mean ± SEM, total n = 7 – 8 mice/group. * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001.
    Figure Legend Snippet: pDC and CD4 - CD8α + DC activation status is influenced by age. The activation status of pDCs and CD4 - CD8α + DCs was evaluated by co-expression of CD40 (A and D) , MHC-I (B and E) and MHC-II (C and F) . Representative data from 1 of 2 experiments is shown as individual mean fluorescent intensities (MFIs) and mean ± SEM, total n = 7 – 8 mice/group. * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001.

    Techniques Used: Activation Assay, Expressing, Mouse Assay

    19) Product Images from "Influenza-Activated ILC1s Contribute to Antiviral Immunity Partially Influenced by Differential GITR Expression"

    Article Title: Influenza-Activated ILC1s Contribute to Antiviral Immunity Partially Influenced by Differential GITR Expression

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00505

    GITR-expression modulates ILC1 functionality upon coculture with H1N1-infected BMDCs. BMDCs generated from wild-type mice using FLT-3 ligand stimulation were infected with the H1N1 PR8 strain (at a MOI of 1). In vitro -generated ILC1s were cocultured overnight with infected or uninfected BMDCs at a 1:1 ratio. The recombinant mouse GITR-Fc chimera protein was applied to the coculture overnight to manipulate GITR expression. Surface and cytokine staining were performed for flow cytometry analysis after 3 h of incubation in media with brefeldin and monensin. (A) GITR expression by ILC1s and BMDCs represented as MFI and representative histogram. (B) MFI of GITR expression by ILC1s with and without GITR-Fc treatment and representative histogram. MFI of (C) IFN-γ, (D) TNF-α, and (E) CD49a expression after GITR-Fc treated ILC1s cocultured with H1N1-infected BMDCs. (F) MFI of CD40, CD80, CD86, and MHC cl. II expression by infected BMDCs post-GITR-Fc treatment. Bars with scatter plots represent the mean ± SEM ( n = 3–4) and MFI data are representative from one out of two independent experiments. Asterisks denote significant values as calculated by nonparametric Mann–Whitney’s test; **** p ≤ 0.0001; *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05.
    Figure Legend Snippet: GITR-expression modulates ILC1 functionality upon coculture with H1N1-infected BMDCs. BMDCs generated from wild-type mice using FLT-3 ligand stimulation were infected with the H1N1 PR8 strain (at a MOI of 1). In vitro -generated ILC1s were cocultured overnight with infected or uninfected BMDCs at a 1:1 ratio. The recombinant mouse GITR-Fc chimera protein was applied to the coculture overnight to manipulate GITR expression. Surface and cytokine staining were performed for flow cytometry analysis after 3 h of incubation in media with brefeldin and monensin. (A) GITR expression by ILC1s and BMDCs represented as MFI and representative histogram. (B) MFI of GITR expression by ILC1s with and without GITR-Fc treatment and representative histogram. MFI of (C) IFN-γ, (D) TNF-α, and (E) CD49a expression after GITR-Fc treated ILC1s cocultured with H1N1-infected BMDCs. (F) MFI of CD40, CD80, CD86, and MHC cl. II expression by infected BMDCs post-GITR-Fc treatment. Bars with scatter plots represent the mean ± SEM ( n = 3–4) and MFI data are representative from one out of two independent experiments. Asterisks denote significant values as calculated by nonparametric Mann–Whitney’s test; **** p ≤ 0.0001; *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05.

    Techniques Used: Expressing, Infection, Generated, Mouse Assay, In Vitro, Recombinant, Staining, Flow Cytometry, Cytometry, Incubation, MANN-WHITNEY

    ILC1s are engaged in cross-talk with DCs during H1N1 infection in vitro . BMDCs generated from wild-type mice using the FLT-3 ligand stimulation were infected with the H1N1 PR8 strain (at a MOI of 1). In vitro -generated ILC1s cultured overnight with infected or uninfected BMDCs at a 1:1 ratio were stained for flow cytometry analysis after 3 h incubation in media with brefeldin and monensin. MFI of ILC1s expressing (A) IFN-γ, (B) TNF-α, (C) GITR, and (D) CD49a upon coculture with H1N1-infected or uninfected BMDCs. (E) Representative histograms for the expression of CD40, CD80, CD86, and MHC cl. II on infected BMDCs. (F) MFI of CD11c + BMDCs expressing CD40, CD80, CD86, and MHC cl. II after coculture with ILC1s. Bars with scatter plots represent the mean ± SEM ( n = 3–4) and MFI data are representative from one out of three independent experiments. Asterisks denote significant values as calculated by nonparametric Kruskal–Wallis test (Dunn’s posttest); **** p ≤ 0.0001; *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05.
    Figure Legend Snippet: ILC1s are engaged in cross-talk with DCs during H1N1 infection in vitro . BMDCs generated from wild-type mice using the FLT-3 ligand stimulation were infected with the H1N1 PR8 strain (at a MOI of 1). In vitro -generated ILC1s cultured overnight with infected or uninfected BMDCs at a 1:1 ratio were stained for flow cytometry analysis after 3 h incubation in media with brefeldin and monensin. MFI of ILC1s expressing (A) IFN-γ, (B) TNF-α, (C) GITR, and (D) CD49a upon coculture with H1N1-infected or uninfected BMDCs. (E) Representative histograms for the expression of CD40, CD80, CD86, and MHC cl. II on infected BMDCs. (F) MFI of CD11c + BMDCs expressing CD40, CD80, CD86, and MHC cl. II after coculture with ILC1s. Bars with scatter plots represent the mean ± SEM ( n = 3–4) and MFI data are representative from one out of three independent experiments. Asterisks denote significant values as calculated by nonparametric Kruskal–Wallis test (Dunn’s posttest); **** p ≤ 0.0001; *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05.

    Techniques Used: Infection, In Vitro, Generated, Mouse Assay, Cell Culture, Staining, Flow Cytometry, Cytometry, Incubation, Expressing

    20) Product Images from "MiR-125b potentiates macrophage activation 1"

    Article Title: MiR-125b potentiates macrophage activation 1

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

    doi: 10.4049/jimmunol.1102001

    MiR-125b increases macrophage response to IFNγ. A) Surface expression of MHCII, CD40, CD86, and CD80 in response to media alone or IFNγ. A representative flow cytometric plot of the IFNγ treated samples is shown for each factor.
    Figure Legend Snippet: MiR-125b increases macrophage response to IFNγ. A) Surface expression of MHCII, CD40, CD86, and CD80 in response to media alone or IFNγ. A representative flow cytometric plot of the IFNγ treated samples is shown for each factor.

    Techniques Used: Expressing, Flow Cytometry

    21) Product Images from "Glycyrrhizin Attenuates Salmonella enterica Serovar Typhimurium Infection: New Insights Into Its Protective Mechanism"

    Article Title: Glycyrrhizin Attenuates Salmonella enterica Serovar Typhimurium Infection: New Insights Into Its Protective Mechanism

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02321

    GL improves surface marker expression in BMDCs. BMDCs were treated with PBS (blank control), GL (200 μg/ml), or LPS (50 ng/ml; positive control) for 48 h. Cells were stained with antibodies against CD40 (A) , CD80 (B) , CD83 (C) , CD86 (D) , and MHC-II (E) , and the fluorescence signals were determined immediately using a FACScan flow cytometer. At least 10,000 events were collected from the cell gate. The data represent three independent experiments.
    Figure Legend Snippet: GL improves surface marker expression in BMDCs. BMDCs were treated with PBS (blank control), GL (200 μg/ml), or LPS (50 ng/ml; positive control) for 48 h. Cells were stained with antibodies against CD40 (A) , CD80 (B) , CD83 (C) , CD86 (D) , and MHC-II (E) , and the fluorescence signals were determined immediately using a FACScan flow cytometer. At least 10,000 events were collected from the cell gate. The data represent three independent experiments.

    Techniques Used: Marker, Expressing, Positive Control, Staining, Fluorescence, Flow Cytometry, Cytometry

    22) Product Images from "Lipid-laden partially-activated plasmacytoid and CD4−CD8α+ dendritic cells accumulate in tissues in elderly mice"

    Article Title: Lipid-laden partially-activated plasmacytoid and CD4−CD8α+ dendritic cells accumulate in tissues in elderly mice

    Journal: Immunity & Ageing : I & A

    doi: 10.1186/1742-4933-11-11

    pDC and CD4 - CD8α + DC activation status is influenced by age. The activation status of pDCs and CD4 - CD8α + DCs was evaluated by co-expression of CD40 (A and D) , MHC-I (B and E) and MHC-II (C and F) . Representative data from 1 of 2 experiments is shown as individual mean fluorescent intensities (MFIs) and mean ± SEM, total n = 7 – 8 mice/group. * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001.
    Figure Legend Snippet: pDC and CD4 - CD8α + DC activation status is influenced by age. The activation status of pDCs and CD4 - CD8α + DCs was evaluated by co-expression of CD40 (A and D) , MHC-I (B and E) and MHC-II (C and F) . Representative data from 1 of 2 experiments is shown as individual mean fluorescent intensities (MFIs) and mean ± SEM, total n = 7 – 8 mice/group. * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001.

    Techniques Used: Activation Assay, Expressing, Mouse Assay

    23) Product Images from "Molecular and functional diversity of distinct subpopulations of extracellular vesicles from stressed pancreatic beta cells: implications for autoimmunity"

    Article Title: Molecular and functional diversity of distinct subpopulations of extracellular vesicles from stressed pancreatic beta cells: implications for autoimmunity

    Journal: bioRxiv

    doi: 10.1101/2020.03.26.003145

    Modulation of co-stimulatory molecules in NOD bmDC by beta AB. NOD Shi mice derived bmDC were incubated for 18h with AB, MV or sEV derived from 8 × 10 7 MIN6 cells. (A) Flow cytometric analysis of the expression of the CD40, MHC II and CD86 activation markers. Histograms with dashed lines represent bmDC treated with AB derived from cytokine beta cells. Histograms with solid lines represent bmDC treated with AB from untreated beta cells. Gray histograms represent isotypic controls. Data from one representative out of 2-5 independent experiments are shown. (B) Scatter plots show the median fluorescence intensity (MFI) of co-stimulatory molecules expression in bmDC after AB treatment normalised to No EV controls (median with range). Tukey’s range test *P
    Figure Legend Snippet: Modulation of co-stimulatory molecules in NOD bmDC by beta AB. NOD Shi mice derived bmDC were incubated for 18h with AB, MV or sEV derived from 8 × 10 7 MIN6 cells. (A) Flow cytometric analysis of the expression of the CD40, MHC II and CD86 activation markers. Histograms with dashed lines represent bmDC treated with AB derived from cytokine beta cells. Histograms with solid lines represent bmDC treated with AB from untreated beta cells. Gray histograms represent isotypic controls. Data from one representative out of 2-5 independent experiments are shown. (B) Scatter plots show the median fluorescence intensity (MFI) of co-stimulatory molecules expression in bmDC after AB treatment normalised to No EV controls (median with range). Tukey’s range test *P

    Techniques Used: Mouse Assay, Derivative Assay, Incubation, Expressing, Activation Assay, Fluorescence

    24) Product Images from "Mapping the SARS-CoV-2 spike glycoprotein-derived peptidome presented by HLA class II on dendritic cells"

    Article Title: Mapping the SARS-CoV-2 spike glycoprotein-derived peptidome presented by HLA class II on dendritic cells

    Journal: bioRxiv

    doi: 10.1101/2020.08.19.255901

    Generation of MDDCs for HLA-II immunopeptidomic profiling. A. Schematic depicting the workflow used to generate the recombinant S protein pulsed MDDC immunopeptidome. MDDCs were pulsed with S or control protein for 18h, and sequential HLA IPs were carried out as indicated. Peptides were eluted from HLA molecules, purified by preparative HPLC and subjected to a LC-MS/MS identification workflow. B. MDDCs were harvested following overnight protein pulse and stained with antibodies to identify DCs and assess their expression of HLA-ABC, HLA-DR, DC-SIGN, DEC-205, CD83, CD40, CD80 and CD86, then analysed by flow cytometry. Histogram plots are gated on live CD11c+CD4+ DCs and show expression of each indicated marker on cells treated with S or control protein. Unstained cells are shown in filled grey histograms. Inset numbers indicate mean fluorescence intensity for each sample.
    Figure Legend Snippet: Generation of MDDCs for HLA-II immunopeptidomic profiling. A. Schematic depicting the workflow used to generate the recombinant S protein pulsed MDDC immunopeptidome. MDDCs were pulsed with S or control protein for 18h, and sequential HLA IPs were carried out as indicated. Peptides were eluted from HLA molecules, purified by preparative HPLC and subjected to a LC-MS/MS identification workflow. B. MDDCs were harvested following overnight protein pulse and stained with antibodies to identify DCs and assess their expression of HLA-ABC, HLA-DR, DC-SIGN, DEC-205, CD83, CD40, CD80 and CD86, then analysed by flow cytometry. Histogram plots are gated on live CD11c+CD4+ DCs and show expression of each indicated marker on cells treated with S or control protein. Unstained cells are shown in filled grey histograms. Inset numbers indicate mean fluorescence intensity for each sample.

    Techniques Used: Recombinant, Purification, High Performance Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Staining, Expressing, Flow Cytometry, Marker, Fluorescence

    Related Articles

    Blocking Assay:

    Article Title: Deficiency of T cell CD40L has minor beneficial effects on obesity-induced metabolic dysfunction
    Article Snippet: .. Blocking CD40L on lymphocytes leads to decreased IL2 production, as well as impaired de novo production of nTregs. .. The CD4CreCD40Lfl/fl mice seem to have a decrease in nTreg development due to T cell CD40L deficiency and reduction in IL2 expression ).

    Article Title: Keratinocytes costimulate naive human T cells via CD2: a potential target to prevent the development of proinflammatory Th1 cells in the skin
    Article Snippet: .. Antibody-mediated blocking of costimulatory molecules In some coculture experiments, blocking antibodies (final concentration: 10 µg/mL) against CD54 (HCD54) (BioLegend, San Diego, CA), CD58 (TS2/9) (BioLegend, San Diego, CA), CD40 (5C3) (BioLegend, San Diego, CA), and CD40L (24–31) (BioLegend, San Diego, CA) and an isotype control (T8E5) (InVivoGen, San Diego, CA) were added to KCs cocultured with naive T cells in serum-free medium (XVIVO-15, Lonza, Basel). .. T cell activation was assessed after 24 h by analyzing the expression of the activation markers CD25 and CD69.

    Expressing:

    Article Title: Programmed death-1 ligands-transfected dendritic cells loaded with glutamic acid decarboxylase 65 (GAD65) inhibit both the alloresponse and the GAD65-reactive lymphocyte response
    Article Snippet: In order to examine if high-level expression of PD-L1 molecules could impact the other phenotype on DC, we tested the expression of CD40, MHC-I, MHC-II molecules, CD80 and CD86 before and after DC transfected with PD-L1. .. No noticeable difference was found among PD-L1/DC, GFP/DC and parental DC for the expression level of CD40, MHC-I and MHC-II molecules ( ). .. However, our results show that DC were 94·8% and 94·5% positive for CD80 and CD86, respectively, and there was no noticeable difference among the above three DC populations for the expression rate of CD80 and CD86.

    Article Title: Comparative Evaluation of αCD40 (2C10R4) and αCD154 (5C8H1 and IDEC-131) in a Nonhuman Primate Cardiac Allotransplant Model
    Article Snippet: .. We hypothesize that the broad distribution and constitutive expression of CD40 on cells of hematopoietic origin may decrease the effective concentration of 2C10R4 in the secondary lymphoid organs and in the graft where “professional” antigen presentation occurs. .. In addition, binding of 2C10R4 to soluble CD40 in plasma may artificially increase reported plasma levels of 2C10R4 to the extent that 2C10R4-CD40 complexes are detected in our assay.

    Article Title: Plasmacytoid dendritic cells and C1q differentially regulate inflammatory gene induction by lupus immune complexes
    Article Snippet: .. After 2 d of stimulation, cells were washed and surface stained to identify monocytes (CD14 clone HCD14, Biolegend, Inc.) and the level of expression of the activation markers CD86 (clone IT2.2, Biolegend, Inc.) and CD40 (clone 5C3, Biolegend Inc.). .. Monocyte subsets were gated for activation analysis as described above.

    Article Title: Myeloid 12/15-LOX regulates B cell numbers and innate immune antibody levelsin vivo
    Article Snippet: However, total CD19+ B cells derived from 12/15-/- mice had altered activation thresholds in comparison to WT, with significantly reduced CD62L expression following LPS stimulation (p ≤ 0.05), similarly Loxoribine and CpG also induced reduced CD62L expression in comparison to WT CD19+ cells. .. Conversely, the expression of CD40 was notably increased with all TLR agonists in 12/15-LOX-/- CD19+ B cells. .. Splenic B cell proliferation is not increased by 12/15-LOX deficiency Since spleen size and total numbers of B1 and Mz B cells are elevated in 12/15-LOX-/- mice, we sought to determine the proliferative capacity of splenic B cells.

    Concentration Assay:

    Article Title: Comparative Evaluation of αCD40 (2C10R4) and αCD154 (5C8H1 and IDEC-131) in a Nonhuman Primate Cardiac Allotransplant Model
    Article Snippet: .. We hypothesize that the broad distribution and constitutive expression of CD40 on cells of hematopoietic origin may decrease the effective concentration of 2C10R4 in the secondary lymphoid organs and in the graft where “professional” antigen presentation occurs. .. In addition, binding of 2C10R4 to soluble CD40 in plasma may artificially increase reported plasma levels of 2C10R4 to the extent that 2C10R4-CD40 complexes are detected in our assay.

    Article Title: Keratinocytes costimulate naive human T cells via CD2: a potential target to prevent the development of proinflammatory Th1 cells in the skin
    Article Snippet: .. Antibody-mediated blocking of costimulatory molecules In some coculture experiments, blocking antibodies (final concentration: 10 µg/mL) against CD54 (HCD54) (BioLegend, San Diego, CA), CD58 (TS2/9) (BioLegend, San Diego, CA), CD40 (5C3) (BioLegend, San Diego, CA), and CD40L (24–31) (BioLegend, San Diego, CA) and an isotype control (T8E5) (InVivoGen, San Diego, CA) were added to KCs cocultured with naive T cells in serum-free medium (XVIVO-15, Lonza, Basel). .. T cell activation was assessed after 24 h by analyzing the expression of the activation markers CD25 and CD69.

    Staining:

    Article Title: Plasmacytoid dendritic cells and C1q differentially regulate inflammatory gene induction by lupus immune complexes
    Article Snippet: .. After 2 d of stimulation, cells were washed and surface stained to identify monocytes (CD14 clone HCD14, Biolegend, Inc.) and the level of expression of the activation markers CD86 (clone IT2.2, Biolegend, Inc.) and CD40 (clone 5C3, Biolegend Inc.). .. Monocyte subsets were gated for activation analysis as described above.

    Activation Assay:

    Article Title: Plasmacytoid dendritic cells and C1q differentially regulate inflammatory gene induction by lupus immune complexes
    Article Snippet: .. After 2 d of stimulation, cells were washed and surface stained to identify monocytes (CD14 clone HCD14, Biolegend, Inc.) and the level of expression of the activation markers CD86 (clone IT2.2, Biolegend, Inc.) and CD40 (clone 5C3, Biolegend Inc.). .. Monocyte subsets were gated for activation analysis as described above.

    Article Title: Distinct CD40L Receptors Mediate Inflammasome Activation and Secretion of IL-1β and MCP-1 in Cultured Human Retinal Pigment Epithelial Cells
    Article Snippet: Our results also demonstrate that a single, circulating endogenous ligand (CD40L) is sufficient to induce inflammasome activation in hRPE cells. .. The secretion of mature IL-1β by hRPE cells was dependent on CD40L binding to α5β1 or CD11b, but not CD40, demonstrating that alternative CD40L receptors may be responsible for downstream inflammasome activation. ..

    other:

    Article Title: Interleukin-21 Drives Proliferation and Differentiation of Porcine Memory B Cells into Antibody Secreting Cells
    Article Snippet: Overall, we have found that inclusion of both APRIL and BAFF on CD40L activated and IL-21 stimulated cells consistently results in numerical enhancement, which is not statistically significant, of cellular viability and antibody secretion as compared to treatment with either one alone.

    Binding Assay:

    Article Title: Distinct CD40L Receptors Mediate Inflammasome Activation and Secretion of IL-1β and MCP-1 in Cultured Human Retinal Pigment Epithelial Cells
    Article Snippet: Our results also demonstrate that a single, circulating endogenous ligand (CD40L) is sufficient to induce inflammasome activation in hRPE cells. .. The secretion of mature IL-1β by hRPE cells was dependent on CD40L binding to α5β1 or CD11b, but not CD40, demonstrating that alternative CD40L receptors may be responsible for downstream inflammasome activation. ..

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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
    https://www.bioz.com/result/anti cd40/product/BioLegend
    Average 86 stars, based on 1 article reviews
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    anti cd40 - by Bioz Stars, 2021-05
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    97
    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
    Price from $9.99 to $1999.99
    anti human cd40 - by Bioz Stars, 2021-05
    97/100 stars
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    PE Dazzle 594 anti human CD40 5C3 Isotype Mouse IgG1 κ Reactivity Human Cross Reactivity Baboon Chimpanzee Cynomolgus Monkey Rhesus Squirrel Monkey Apps FC Size 25 tests
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    Image Search Results


    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