16-0409-81

antibody from Invitrogen Antibodies

Targeting: CD40 Bp50, p50, TNFRSF5

Provider product page for 16-0409-81

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [19]
Comments [0]
Validations
Flow cytometry [1]
Other assay [9]

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Product number: 16-0409-81 - 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

CD40 protein structure - 16-0409-81 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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.

Supportive validation

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Experimental details: 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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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