Antibody data
- Antibody Data
- Antigen structure
- References [18]
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- Validations
- Flow cytometry [1]
- Other assay [9]
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- Product number
- 16-0409-025 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- CD40 Monoclonal Antibody (5C3), Functional Grade, eBioscience™
- Antibody type
- Monoclonal
- Antigen
- Other
- Description
- Description: The 5C3 monoclonal antibody reacts with human CD40, a 45-50 kDa type I transmembrane glycoprotein. CD40 is a member of the TNFR family and is expressed by B lymphocytes, follicular dendritic cells, thymic epithelium, and a subset of peripheral T cells. CD40 regulates B cell development and maturation by inducing Ig isotype-switching and in combination with other signals such as IL-4, protects B cells from surface Ig-induced apoptosis and promotes proliferation. Interaction of CD40 with CD154 (gp39), its ligand on T cells, is important in T-B cell crosstalk and plays a role in costimulation and immune regulation. 5C3 is reported to be used for activation of B cells in in vitro functional assays.
- Antibody clone number
- 5C3
- Concentration
- 1 mg/mL
Submitted references Recombinant human IL-37 inhibited endometriosis development in a mouse model through increasing Th1/Th2 ratio by inducing the maturation of dendritic cells.
Activated monocytes and markers of inflammation in newly diagnosed multiple sclerosis.
A replication-incompetent CD154/40L recombinant vaccinia virus induces direct and macrophage-mediated antitumor effects in vitro and in vivo.
Transitional B Cells and TLR9 Responses Are Defective in Selective IgA Deficiency.
Subsets of activated monocytes and markers of inflammation in incipient and progressed multiple sclerosis.
Comprehensive characterization of chorionic villi-derived mesenchymal stromal cells from human placenta.
An Immune Atlas of Clear Cell Renal Cell Carcinoma.
Surrogate in vitro activation of innate immunity synergizes with interleukin-7 to unleash rapid antigen-driven outgrowth of CD4+ and CD8+ human peripheral blood T-cells naturally recognizing MUC1, HER2/neu and other tumor-associated antigens.
CD16(+) Monocyte Subset Was Enriched and Functionally Exacerbated in Driving T-Cell Activation and B-Cell Response in Systemic Lupus Erythematosus.
A simple, accurate and universal method for quantification of PCR.
Transcriptional and functional characterization of CD137L-dendritic cells identifies a novel dendritic cell phenotype.
Direct tumor recognition by a human CD4(+) T-cell subset potently mediates tumor growth inhibition and orchestrates anti-tumor immune responses.
A novel subset of B7-H3(+)CD14(+)HLA-DR(-/low) myeloid-derived suppressor cells are associated with progression of human NSCLC.
Endothelial microparticles interact with and support the proliferation of T cells.
A CD8 T cell/indoleamine 2,3-dioxygenase axis is required for mesenchymal stem cell suppression of human systemic lupus erythematosus.
Severe loss of invariant NKT cells exhibiting anti-HTLV-1 activity in patients with HTLV-1-associated disorders.
CD46-induced human Treg enhance B-cell responses.
Airway epithelial cells produce B cell-activating factor of TNF family by an IFN-beta-dependent mechanism.
Li L, Liao Z, Ye M, Jiang J
Reproductive biology and endocrinology : RB&E 2021 Aug 24;19(1):128
Reproductive biology and endocrinology : RB&E 2021 Aug 24;19(1):128
Activated monocytes and markers of inflammation in newly diagnosed multiple sclerosis.
Carstensen M, Christensen T, Stilund M, Møller HJ, Petersen EL, Petersen T
Immunology and cell biology 2020 Aug;98(7):549-562
Immunology and cell biology 2020 Aug;98(7):549-562
A replication-incompetent CD154/40L recombinant vaccinia virus induces direct and macrophage-mediated antitumor effects in vitro and in vivo.
Governa V, Brittoli A, Mele V, Pinamonti M, Terracciano L, Muenst S, Iezzi G, Spagnoli GC, Zajac P, Trella E
Oncoimmunology 2019;8(5):e1568162
Oncoimmunology 2019;8(5):e1568162
Transitional B Cells and TLR9 Responses Are Defective in Selective IgA Deficiency.
Lemarquis AL, Einarsdottir HK, Kristjansdottir RN, Jonsdottir I, Ludviksson BR
Frontiers in immunology 2018;9:909
Frontiers in immunology 2018;9:909
Subsets of activated monocytes and markers of inflammation in incipient and progressed multiple sclerosis.
Gjelstrup MC, Stilund M, Petersen T, Møller HJ, Petersen EL, Christensen T
Immunology and cell biology 2018 Feb;96(2):160-174
Immunology and cell biology 2018 Feb;96(2):160-174
Comprehensive characterization of chorionic villi-derived mesenchymal stromal cells from human placenta.
Ventura Ferreira MS, Bienert M, Müller K, Rath B, Goecke T, Opländer C, Braunschweig T, Mela P, Brümmendorf TH, Beier F, Neuss S
Stem cell research & therapy 2018 Feb 5;9(1):28
Stem cell research & therapy 2018 Feb 5;9(1):28
An Immune Atlas of Clear Cell Renal Cell Carcinoma.
Chevrier S, Levine JH, Zanotelli VRT, Silina K, Schulz D, Bacac M, Ries CH, Ailles L, Jewett MAS, Moch H, van den Broek M, Beisel C, Stadler MB, Gedye C, Reis B, Pe'er D, Bodenmiller B
Cell 2017 May 4;169(4):736-749.e18
Cell 2017 May 4;169(4):736-749.e18
Surrogate in vitro activation of innate immunity synergizes with interleukin-7 to unleash rapid antigen-driven outgrowth of CD4+ and CD8+ human peripheral blood T-cells naturally recognizing MUC1, HER2/neu and other tumor-associated antigens.
Pathangey LB, McCurry DB, Gendler SJ, Dominguez AL, Gorman JE, Pathangey G, Mihalik LA, Dang Y, Disis ML, Cohen PA
Oncotarget 2017 Feb 14;8(7):10785-10808
Oncotarget 2017 Feb 14;8(7):10785-10808
CD16(+) Monocyte Subset Was Enriched and Functionally Exacerbated in Driving T-Cell Activation and B-Cell Response in Systemic Lupus Erythematosus.
Zhu H, Hu F, Sun X, Zhang X, Zhu L, Liu X, Li X, Xu L, Shi L, Gan Y, Su Y
Frontiers in immunology 2016;7:512
Frontiers in immunology 2016;7:512
A simple, accurate and universal method for quantification of PCR.
Boulter N, Suarez FG, Schibeci S, Sunderland T, Tolhurst O, Hunter T, Hodge G, Handelsman D, Simanainen U, Hendriks E, Duggan K
BMC biotechnology 2016 Mar 9;16:27
BMC biotechnology 2016 Mar 9;16:27
Transcriptional and functional characterization of CD137L-dendritic cells identifies a novel dendritic cell phenotype.
Harfuddin Z, Dharmadhikari B, Wong SC, Duan K, Poidinger M, Kwajah S, Schwarz H
Scientific reports 2016 Jul 19;6:29712
Scientific reports 2016 Jul 19;6:29712
Direct tumor recognition by a human CD4(+) T-cell subset potently mediates tumor growth inhibition and orchestrates anti-tumor immune responses.
Matsuzaki J, Tsuji T, Luescher IF, Shiku H, Mineno J, Okamoto S, Old LJ, Shrikant P, Gnjatic S, Odunsi K
Scientific reports 2015 Oct 8;5:14896
Scientific reports 2015 Oct 8;5:14896
A novel subset of B7-H3(+)CD14(+)HLA-DR(-/low) myeloid-derived suppressor cells are associated with progression of human NSCLC.
Zhang G, Huang H, Zhu Y, Yu G, Gao X, Xu Y, Liu C, Hou J, Zhang X
Oncoimmunology 2015 Feb;4(2):e977164
Oncoimmunology 2015 Feb;4(2):e977164
Endothelial microparticles interact with and support the proliferation of T cells.
Wheway J, Latham SL, Combes V, Grau GE
Journal of immunology (Baltimore, Md. : 1950) 2014 Oct 1;193(7):3378-87
Journal of immunology (Baltimore, Md. : 1950) 2014 Oct 1;193(7):3378-87
A CD8 T cell/indoleamine 2,3-dioxygenase axis is required for mesenchymal stem cell suppression of human systemic lupus erythematosus.
Wang D, Feng X, Lu L, Konkel JE, Zhang H, Chen Z, Li X, Gao X, Lu L, Shi S, Chen W, Sun L
Arthritis & rheumatology (Hoboken, N.J.) 2014 Aug;66(8):2234-45
Arthritis & rheumatology (Hoboken, N.J.) 2014 Aug;66(8):2234-45
Severe loss of invariant NKT cells exhibiting anti-HTLV-1 activity in patients with HTLV-1-associated disorders.
Azakami K, Sato T, Araya N, Utsunomiya A, Kubota R, Suzuki K, Hasegawa D, Izumi T, Fujita H, Aratani S, Fujii R, Yagishita N, Kamijuku H, Kanekura T, Seino K, Nishioka K, Nakajima T, Yamano Y
Blood 2009 Oct 8;114(15):3208-15
Blood 2009 Oct 8;114(15):3208-15
CD46-induced human Treg enhance B-cell responses.
Fuchs A, Atkinson JP, Fremeaux-Bacchi V, Kemper C
European journal of immunology 2009 Nov;39(11):3097-109
European journal of immunology 2009 Nov;39(11):3097-109
Airway epithelial cells produce B cell-activating factor of TNF family by an IFN-beta-dependent mechanism.
Kato A, Truong-Tran AQ, Scott AL, Matsumoto K, Schleimer RP
Journal of immunology (Baltimore, Md. : 1950) 2006 Nov 15;177(10):7164-72
Journal of immunology (Baltimore, Md. : 1950) 2006 Nov 15;177(10):7164-72
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Supportive validation
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- Staining of normal human peripheral blood cells with Anti-Human CD40 FITC (left) or PE (right).Appropriate isotype controls were used (open histogram).Cells in the lymphocyte population were used for analysis.
Supportive validation
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- Gating strategy used in the flow cytometric analysis of patient and healthy control samples. A sample from a representative patient with RRMS was used for this figure. (a) From left to right: Total monocytes (> 20,000 events) were gated according to their size and granularity in forward scatter-height ( FSC -H)/side scatter-height ( SSC -H), aggregated cells were removed according to forward scatter-area ( FSC -A)/ FSC -H and side scatter-area ( SSC -A)/ SSC -H, and finally dead cells were removed according to staining with a LIVE / DEAD (r) cell stain. (b) From left to right: The tree monocyte subsets (classical, intermediate, nonclassical) were gated on the ""Live Cells"" gate, as were the CD 40+, CD 163+, and CD 192+ cells (blue). Appropriate isotype controls (red) were used to determine the unspecific antibody binding. (c, d) From left to right: Human endogenous retrovirus ( HERV ) expression was determined on the total monocyte population (Live cells) and the three monocyte subsets (classical, intermediate, nonclassical) by incubation with sera from rabbits immunized with HERV H3 Env (c) or HERV W3 Env (d) peptide antigens (blue) as described previously and with the appropriate control (pre-immune sera) (red) to determine the median fluorescence index (MFI).
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- Fig. 3 Quantification of CD40 , IL7R and GAPDH in PBMCs stimulated with 0-1x PMA/ionomycin. a Absolute quantification of CD40 , IL7R and GAPDH in PBMCs stimulated with 0, 0.25x, 0.5x and 1x PMA/ionomycin (20 ng ml -1 PMA, 500 ng ml -1 ionomycin; PMA/I) by RealCount software following qPCR using AccuCal-D calibrators. b Relative expression levels of CD40 and IL7R in PBMCs stimulated with 0, 0.25x, 0.5x and 1x PMA/ionomycin (20 ng ml -1 PMA, 500 ng ml -1 ionomycin). The hatched bars are relative expression levels determined by DeltaDeltaCq using GAPDH as the reference gene and no PMA/ionomycin as the control sample, solid bars are relative expression levels determined by Pfaffl analysis, using GAPDH as reference gene, unstimulated cells as controls and individual efficiency values calculated by RealCount software, and the checkered bars are quantified by RealCount software following inclusion of AccuCal-D in the same PCR run, and expressed relative to the no PMA/ionomycin control. c Representative overlay graphs from flow cytometry showing relative measurement of CD40 and IL7R in the same population of PBMCs stimulated with 0 ( red ), 0.25x ( blue ), 0.5x ( green ) and 1x PMA/ionomycin (20 ng ml -1 PMA, 500 ng ml -1 ionomycin; orange ) as in ( a ). **** p < 0.0001 relative to respective no PMA/ionomycin control, n = 4
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- Figure S1 Comparison of FACS and Mass Cytometry Analyses, Related to Figure 1 (A) Scheme illustrating the in vitro system used to generate the monocyte-derived macrophage (MDM) populations from monocytes isolated from the blood of healthy donors. (B) Heatmap showing the mean expression of the indicated surface markers among the different MDMs and blood monocyte populations. Only markers showing a 2-fold change among the MDM populations were included. (C) Correlations between FACS and mass cytometry measurements for the 33 antibodies present both in the mass cytometry antibody panel and in the cell surface flow cytometry antibody screen were assessed using a linear regression model. The coefficient of determination R 2 and the linear model are shown for each antibody. MFI, mean fluorescent intensity; MC, metal count.
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- Figure 1 Gating strategy used in the flow cytometric analysis. A sample from a representative patient with RRMS was used for this figure. (a) From left to right: Total PBMCs (> 300,000 events) were gated according to their size and granularity in forward scatter-area (FSC-A) / side scatter-area (SSC-A); aggregated cells were removed according to forward scatter-area (FSC-A) / FSC-H and side scatter-area (SSC-A) / SSC-H leaving only singlets; dead cells as well as NK-cells were removed according to staining with a LIVE/DEAD cell stain and monoclonal anti-CD56 antibody, respectively; monocytes (> 100,000 events) were gated in forward scatter-area (FSC-A) / side scatter-area (SSC-A); and finally the three monocyte subsets (classical, intermediate, non-classical) were gated according to their CD14/CD16 expression. (b) From left to right: Surface marker expression of CD11b, CD18, CD40, CD64, CD86, CD163, CCR1, CCR2, CCR5, and TACE were determined by gating on positive cells (blue). Appropriate isotype controls (red) were used to determine the unspecific antibody binding. (c) From left to right: Human endogenous retrovirus (HERV) expression was determined on the monocyte population, and on the three monocyte subsets (classical, intermediate, non-classical) by incubation with sera from rabbits immunized with HERV H3 Env (top row) or HERV W3 Env (bottom row) peptide antigens (blue) as described previously 36 and with the appropriate control (pre-immune sera) (red) to determine the po
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- Figure 2 Differences in the expression of CD11b, CD18, CD40, CD64, CD86, CD163, CCR1, CCR2, CCR5 and TACE on the monocytes. The differences in expression of CD11b, CD18, CD40, CD64, CD86, CD163, CCR1, CCR2, CCR5 and TACE on the monocyte population (Monocytes, Figure 1a ) from the five patient groups were determined as the percentage of positive cells. Bars represent the median of the populations. CIS, clinically isolated syndrome; PMS, progressive multiple sclerosis; Pt, patients; RIS, radiologically isolated syndrome; RRMS, relapsing-remitting multiple sclerosis; SC, symptomatic control.
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- Fig. 7 rhIL-37-induced the increasing of CD40, CD80, CD86, and MHC II in DCs was rescued by activation of STAT3. (A-D) The percentages of CD40-, CD80-, CD86-, and MHC II-positive DCs were determined using flow cytometry. N = 3. ** P < 0.01 compared with Control, and ## P < 0.01 compared with Control + rhIL-37
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- Figure 6 Exposure to R848 and LPS is a critical determinant of CD8+ T-cell sensitization A . left panel. IL-12 and IL-23 production during first two days of culture are both markedly enhanced by dual stimulation with R848 and LPS. Methods as in Figure 1B . Note that cytokine production is shown in log scale. This graphing of ELISA results shows an averaging of 4 biological replicates. A . right panel, exposure to R848+LPS enhances frequency and/or density of co-stimulatory ligands and receptors on PBMC CD33+ constituents. Methods as in Figure 1B ., with FACS analyses performed after 48h in culture. Frequency of each molecule of interest in the CD33+ subpopulation was determined and averaged for 4 biological replicates; in addition, fold-increase in cell surface staining density relative to isotype control (averaged Mean Fluorescent Index) was determined. Frequency of expression is shown graphically, whereas for MFI only tabulated p values are shown. Exposure to R848+LPS significantly increased both the frequency and MFI of CD70, 4-1BB ligand, and B7.1 (CD80) (shown on graph). In addition, although frequency of CD40 expression was already nearly uniform without R848+LPS exposure, R848+LPS significantly increased the MFI for CD40. Two-tailed p values are shown, based on a paired Student t -test comparing with and without R848+LPS exposure within each experimental run. B, left panel, dual exposure to R848+LPS significantly enhances Ag-specific CD4+ and CD8+ propagation. Cultures
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- Fig. 2 a Cumulative population-doubling (cPD) levels versus passage number for the four different sources of MSC. Black represents CV-MSC ( n = 7), dark gray UC-MSC ( n = 4), medium gray AT-MSC ( n = 5), and light gray BM-MSC ( n = 6). b IHC-based senescence-associated beta-galactosidase (SA-beta-gal) staining of CV-MSC in early (i, passage 4) and late (ii, passage 9) passages, AT-MSC in passage 6 (iii), BM-MSC in passage 6 (iv), and UC-MSC in passage 2 (v) and passage 4 (vi). Scale = 200 mum. c IHC of CV-MSC (i, ii) and BM-MSC (iii, iv) stained for osteopontin (i, iii) and fibronectin (ii, iv). Scale = 1 mm. d Collagen area (%) after collagen contraction assay for CV-MSC ( n = 4), BM-MSC ( n = 3), UC-MSC ( n = 4), and AT-MSC ( n = 3). Cells in passage 3 were used. Results expressed as mean +- SD, percentage of the total collagen area of the collagen gels without cells. e Surface marker expression of CV-MSC in early passages ( n = 5). Results expressed as mean +- SD (%). f Representative immunofluorescence of early passaged CV-MSC (i, iii) and BM-MSC (iii, iv) stained for SM22alpha (i, iii) and alpha-SMA (ii, iv). Scale = 50 mum. AT adipose tissue, BM bone marrow, CV chorionic villi, MSC mesenchymal stromal cells, UC umbilical cord