CD40 antibody | 3/23
Rat anti Mouse CD40 antibody, clone 3/23 (MCA1143) used for the evaluation of CD40 expression by microglia in vitro following treatment with ifnγ or LPS by immunofluorescence and western blotting.
Image caption:
Elevated CD40 expression by LPS or IFNγ in the BV-2 and primary microglia cells is regulated by cPLA2α.
A representative double-immunofluorescence staining of cPLA2α (green) and CD40 (red) in unstimulated or stimulated microglia by LPS or IFNγ in the absence or presence of AS or sense (SE). DAPI staining shows cell nuclei. The BV-2 cells were treated with (a) 50 ng/ml LPS or (b) 10 ng/ml IFNγ for 24 h.
Four micrometer AS or the corresponding sense (SE) were added to the cultures 24 h before addition of the stimuli. The intensity of CD40 or cPLA2α were quantitated for the cell and expressed in the bar graph as arbitrary units. A representaive immunoblot analysis of cPLA2α and CD40 for the BV-2 microglia cells (c, d) treated as (a) and (b), The intensity of each cPLA2α or CD40 band after quantification by densitometry was divided by the intensity of each calreticulin (Calre) band and expressed as arbitrary units. The bar graphs are the mean ± SE from three independent experiments (***p<0.0001).
From:Malada-Edelstein YF, Hadad N, Levy R.
Regulatory role of cytosolic phospholipase A(2) alpha in the induction of CD40 in microglia.
J Neuroinflammation. 2017 Feb 10;14(1):33..
This image is from an open access article distributed under terms of a Creative Commons Attribution License.
Rat anti Mouse CD40 antibody, clone 3/23 used for the evaluation of CD40 expression by microglia in vitro following treatment with ifnγ or LPS by immunofluorescence and western blotting.
Image caption:
Elevated CD40 expression by LPS or IFNγ in the BV-2 and primary microglia cells is regulated by cPLA2α.
A representative double-immunofluorescence staining of cPLA2α (green) and CD40 (red) in unstimulated or stimulated microglia by LPS or IFNγ in the absence or presence of AS or sense (SE). DAPI staining shows cell nuclei. The mouse primary microglia cells were treated with (e) 50 ng/ml LPS or (f) 25 ng/ml IFNγ for 48 h. Scale bars = 50 μm. A representaive immunoblot analysis of cPLA2α and CD40 for the primary mouse microglia (g, h) treated as (e), and (f).
Four micrometer AS or the corresponding sense (SE) were added to the cultures 24 h before addition of the stimuli. The intensity of CD40 or cPLA2α were quantitated for the cell and expressed in the bar graph as arbitrary units. The bar graphs are the mean ± SE from three independent experiments (***p<0.0001).
From: Malada-Edelstein YF, Hadad N, Levy R.
Regulatory role of cytosolic phospholipase A(2) alpha in the induction of CD40 in microglia.
J Neuroinflammation. 2017 Feb 10;14(1):33..
This image is from an open access article distributed under terms of a Creative Commons Attribution License.
Rat anti Mouse CD40 antibody, clone 3/23 used for the evaluation of CD40 expression by microglia in vitro following treatment with ifnγ or LPS by immunofluorescence and western blotting.
Image caption:
cPLA2α activates NFκB through activation of NOX2-NADPH oxidase in the BV-2 microglia cells under LPS stimulation. a A representative immunoblot analysis of the kinetics of cPLA2α phosphorylation induced by 50 ng/ml LPS, out of three independent experiments. The intensity of each phosphorylated cPLA2α (p-cPLA2α Ser-505) band after quantification by densitometry was divided by the intensity of each cPLA2α band and expressed as arbitrary units. The bar graphs are the mean ± SE from three independent experiments. b The BV-2 cells were treated with 2 μM pyrrophenone (Pyrro) or 5 μM DPI for 60 min before stimulation with 50 ng/ml LPS for 15 min. The intensity of phosphorylated cPLA2α was quantitated by densitometry as described in (a). The bar graphs are the mean ± SE from three independent experiments. c The effect of cPLA2α inhibition on superoxide production in the unstimulated or stimulated BV-2 cells with 50 ng/ml LPS for 15 min was detected by DHE reduction. Two micrometer pyropheonoe (Pyrro) or 200 μm apocynin (used as a positive control) were added to the cells 60 min before stimulation with LPS. AS or sense were added 24 h prior to addition of LPS. DAPI staining shows cell nuclei. Scale bars large = 50μm, insert = 20μm. The intensity of reduced DHE was quantitated and expressed in the bar graph as arbitrary units. The bar graphs are the mean ± SE from three independent experiments. d Immunoprecipitation of p47phox and phoshpo cPLA2α (pcPLA2α) in the membrane fraction of unstimulated microglia and stimulated with LPS for 15 min. Shown a representative immunoblot of three experiments. e Addition of 10 μM arachidonic acid together with LPS to cells pretreated for 24 h with antisense against cPLA2α restored the expression of CD40 protein. Shown a representative immunofluorescence staining of CD40. DAPI staining shows cell nuclei. The intensity of CD40 was quantitated for the cell and expressed in the bar graph as arbitrary units. Scale bars = 50μm. The bar graph is the mean ± SE from three independent experiments. (***p<0.0001). f FACS analysis of CD40 protein expression in the unstimulated or stimulated BV-2 cells with 50 ng/ml LPS for 24 h in the absence or presence of 2μM pyrrophenone or 5μM DPI (added to the cells 60 min before stimulation with LPS). The bar graphs are the X-median ± SE from five independent experiments. g A representative immunoblot analysis of NFκB p-65 phosphorylation (p-NFκB p-65 Ser-536) in unstimulated or stimulated BV-2 microglia by 50 ng/ml LPS for 15 min in the absence or presence of 2μM pyrrophenone or 5μM DPI. The intensity of each phosphorylated NFκB p-65 band after quantification by densitometry was divided by the intensity of each NFκB p-65 band and expressed as arbitrary units. The bar graphs are the mean ± SE from three independent experiments. h A representative immunoblot analysis of phoshpo cPLA2α and phospho NFκB p-65 subunit and phosspho ERK1/2 in unstimulated or stimulated BV-2 microglia by 50 ng/ml LPS for 15 min in the absence or presence of 5μM OU126. The bar graphs are the mean ± SE of the intensity of the quantitated phosphorylated forms divided by the non-phophorylated forms of three independent experiments. i The involvement of TRIF and MyD88 pathways in activation of cPLA2α in the signaling leading to CD40 upregualtion. The BV-2 cells were incubated with TRIF or MyD88 peptide inhibitors for 60 min before stimulation with 50 ng/ml LPS for 15 min. A representative immunoblot analysis of cPLA2α activity, out of three independent experiments is presented. The intensity of phosphorylated cPLA2α (p-cPLA2α Ser-505) was quantitated by densitometry as described in A. j Shown is a representative immunofluorescence analysis of CD40 protein expression in the cells treated as in i. DAPI staining shows cell nuclei. Scale bars = 50μm. The intensity of cPLA2α and of CD40 was quantitated and expressed in the bar graph as arbitrary units. The bar graph is the mean ± SE from three independent experiments. (***p<0.0001,**p<0.001, n.s. not significant).
From: Malada-Edelstein YF, Hadad N, Levy R.
Regulatory role of cytosolic phospholipase A(2) alpha in the induction of CD40 in microglia.
J Neuroinflammation. 2017 Feb 10;14(1):33..
This image is from an open access article distributed under terms of a Creative Commons Attribution License.
Rat anti Mouse CD40 antibody, clone 3/23 used for the evaluation of CD40 expression by microglia in vitro following treatment with ifnγ or LPS by immunofluorescence.
Image caption:
Endogenously produced TNF-α regulates cPLA2&α activation in the BV2 cells under IFNγ stimulation.
Immunofluorescence analysis of CD40 protein expression in unstimulated or stimulated BV-2 microglia with 10 ng/ml IFNγ for 24 h in the absence or presence of 50 ng/ml TNF-α-neutralizing antibody or stimulated by 10 ng/ml TNF-α. Scale bars = 50μm. The intensity of CD40 was quantitated and expressed in the bar graph as arbitrary units. The bar graph is the mean ± SE from four independent experiments. DAPI staining shows cell nuclei. (***p<0.0001, n.s. not significant)
From: Malada-Edelstein YF, Hadad N, Levy R.
Regulatory role of cytosolic phospholipase A(2) alpha in the induction of CD40 in microglia.
J Neuroinflammation. 2017 Feb 10;14(1):33..
This image is from an open access article distributed under terms of a Creative Commons Attribution License.
Rat anti Mouse CD40
- Product Type
- Monoclonal Antibody
- Clone
- 3/23
- Isotype
- IgG2a
- Specificity
- CD40
Antibody Quality and Performance Guarantee
| Rat anti Mouse CD40 antibody, clone 3/23 recognizes the murine CD40 cell surface glycoprotein. It does not react with normal mouse Ig or with human IgG1 and will stain most mature mouse B cells. It does not cross react with mouse T cells. The specificity of Rat anti Mouse CD40 antibody, clone 3/23 was demonstrated by ELISA and flow cytometry using BHK cells transfected with mouse CD40. Rat anti Mouse CD40 antibody, clone 3/23 is a powerful activator of normal B cells especially in the presence of IL-4. |
- Target Species
- Mouse
- Product Form
- Purified IgG - liquid
- Preparation
- Purified IgG prepared by affinity chromatography on Protein G from tissue culture supernatant
- Buffer Solution
- Phosphate buffered saline
- Preservative Stabilisers
- 0.09% sodium azide (NaN3)
- Carrier Free
- Yes
- Immunogen
- Extracellular Domain of Mouse CD40 and the Fc portion of Human IgG1.
- Approx. Protein Concentrations
- IgG concentration 1 mg/ml
- Fusion Partners
- Spleen cells from immunised LOU/c rats were fused with cells of the Ag8 mouse myeloma cell line.
- Regulatory
- For research purposes only
- Guarantee
- 12 months from date of despatch
This product is shipped at ambient temperature. It is recommended to aliquot and store at -20°C on receipt. When thawed, aliquot the sample as needed. Keep aliquots at 2-8°C for short term use (up to 4 weeks) and store the remaining aliquots at -20°C.
Avoid repeated freezing and thawing as this may denature the antibody. Storage in frost-free freezers is not recommended.
Avoid repeated freezing and thawing as this may denature the antibody. Storage in frost-free freezers is not recommended.
This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visit the antibody protocols page.
| Application Name | Verified | Min Dilution | Max Dilution |
|---|---|---|---|
| ELISA |  |
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| Flow Cytometry | |
1/10 Pack Size: 0.1 mg 1/50 Pack Size: 0.25 mg |
1/50 Pack Size: 0.1 mg 1/100 Pack Size: 0.25 mg |
| Immunofluorescence | |
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| Immunohistology - Frozen | |
Where this antibody has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the antibody for use in their own system using appropriate negative/positive controls.
- Flow Cytometry
- Use 10μl of the suggested working dilution to label 106 cells in 100μl.
| Description | Product Code | Applications | Pack Size | List Price | Your Price | Quantity | |
|---|---|---|---|---|---|---|---|
| Rat IgG2a Negative Control | MCA1212 | E F | 1 ml |
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Log in | ||
| List Price | Your Price | ||||||
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| Description | Rat IgG2a Negative Control | ||||||
Source Reference
-
Hasbold, J. et al. (1994) Properties of mouse CD40: cellular distribution of CD40 and B cell activation by monoclonal anti-mouse CD40 antibodies.
Eur J Immunol. 24 (8): 1835-42.
References for CD40 antibody
-
Parry, S.L. et al. (1994) Plastic-immobilized anti-mu or anti-delta antibodies induce apoptosis in mature murine B lymphocytes.
Eur J Immunol. 24 (4): 974-9. -
Hasbold, J. & Klaus, G.G. (1994) B cells from CBA/N mice do not proliferate following ligation of CD40.
Eur J Immunol. 24 (1): 152-7. -
Salomon, B. et al. (1998) Three populations of mouse lymph node dendritic cells with different origins and dynamics.
J Immunol. 160: 708-17. -
Yang, Y.F. et al. (2000) Requirement for IFN-gamma in IL-12 production induced by collaboration between v(alpha)14(+) NKT cells and antigen-presenting cells.
Int Immunol. 12: 1669-75. -
Ogasawara, K. et al. (2002) Profiles of cell-to-cell interaction of Mycobacterium intracellulare-induced immunosuppressive macrophages with target T cells in terms of suppressor signal transmission.
Clin Exp Immunol. 129: 272-80. -
Russo, S. et al. (2003) Platelet-activating factor mediates CD40-dependent angiogenesis and endothelial-smooth muscle cell interaction.
J Immunol. 171: 5489-97. -
Mukhopadhyay, S. et al. (2004) Activation of murine macrophages by Neisseria meningitidis and IFN-gamma in vitro: distinct roles of class A scavenger and Toll-like pattern recognition receptors in selective modulation of surface phenotype.
J Leukoc Biol. 76: 577-84. -
Mohan, J. et al. (2005) Skin-derived dendritic cells acquire and degrade the scrapie agent following in vitro exposure.
Immunology. 116: 122-33.
View The Latest Product References
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Bedoret, D. et al. (2009) Lung interstitial macrophages alter dendritic cell functions to prevent airway allergy in mice.
J Clin Invest. 119 (12): 3723-38. -
Shakib, S. et al. (2009) Checkpoints in the development of thymic cortical epithelial cells.
J Immunol. 182: 130-7. -
Legutko, A. et al. (2011) Sirtuin 1 Promotes Th2 Responses and Airway Allergy by Repressing Peroxisome Proliferator-Activated Receptor-{gamma} Activity in Dendritic Cells.
J Immunol. 187: 4517-29. -
Pletinckx, K. et al. (2015) Immature dendritic cells convert anergic nonregulatory T cells into Foxp3- IL-10+ regulatory T cells by engaging CD28 and CTLA-4.
Eur J Immunol. 45 (2): 480-91. -
Malada-Edelstein, Y.F. et al. (2017) Regulatory role of cytosolic phospholipase A2 alpha in the induction of CD40 in microglia.
J Neuroinflammation. 14 (1): 33.
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