CD154 Antibody, anti-human, REAfinity™

Name: CD154 Antibody, anti-human, REAfinity™
Availability: InStock

CD154 Antibody, anti-human, REAfinity™Extended ValidationRecombinant

Clone:

REA238

Type of antibody:

Primary antibodies, Recombinant antibodies

Isotype:

recombinant human IgG1

Applications:

FC

Alternative names:

CD40L, CD40-L, gp39, T-BAM, TRAP, Ly-62, HIGM1, IGM

Intracellular flow cytometry applications forCD154 Antibody, anti-human, REAfinity™

APC
APC-Vio 770
Biotin
FITC
PE
PE-Vio 615
PE-Vio 770
PerCP-Vio 700
Vio Bright B515
VioBlue

Conjugate:

APC

Recommended dilution:

1:50

Remark:

Cells should be stained prior to fixation, if formaldehyde is used as a fixative.

Tested:

QC tested

Products used:

130-114-136, 130-113-610

Protocols:

Cell surface flow cytometry staining protocol (PBS/EDTA/BSA 1:50)

Description:

Human peripheral blood mononuclear cells (PBMCs), either left unstimulated (left images) or stimulated with CytoStim™ and 1 µg/mL CD40 for 16 hours, were stained with CD154 antibodies as well as with CD4 antibodies. Cells were analyzed by flow cytometry using the MACSQuant^® Analyzer. The Tandem Signal Enhancer has been used to increase binding specificity of tandem-dye–conjugated antibodies. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence or 4',6-diamidino-2-phenylindole (DAPI) fluorescence, as in the case of tandem conjugates.

Extended validation for CD154 Antibody, anti-human, REAfinity™

Specificity

Epitope competition

In order to compare the epitope specificity of an antibody, the clone being used is compared with other known clones recognizing the same antigen in a competition assay.

Other clones	Overlap in epitope recognition with REA238
24-31	++
5C8	++
TRAP1	+
Cells were incubated with an excess of purified unconjugated CD154 (REA238) antibody followed by staining with fluorochrome-conjugated antibodies of other known clones against the same marker. Based on the fluorescence signal obtained, the clones were identified as recognizing completely overlapping (++), partially overlapping (+), or completely different epitopes (-) of the marker.

Sensitivity

Performance comparison

Selected fluorochrome conjugated antibodies from Miltenyi Biotec were compared to commercially available hybridoma clones in flow cytometry analysis.

Flow cytometric comparison of different clones for CD154. Human peripheral blood mononuclear cells (PBMCs), stimulated with 1 µg/mL SEB and 1 µg/mL CD40 for 6 hours, were stained with CD154 antibodies and with a suitable counterstaining. As a control, CD154 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Flow cytometric comparison of different clones for CD154. Human peripheral blood mononuclear cells (PBMCs), stimulated with 1 µg/mL SEB and 1 µg/mL CD40 for 6 hours, were stained with CD154 antibodies and with a suitable counterstaining. As a control, CD154 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Flow cytometric comparison of different clones for CD154. Human peripheral blood mononuclear cells (PBMCs), stimulated with 1 µg/mL SEB and 1 µg/mL CD40 for 6 hours, were stained with CD154 antibodies and with a suitable counterstaining. As a control, CD154 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Flow cytometric comparison of different clones for CD154. Human peripheral blood mononuclear cells (PBMCs), stimulated with 1 µg/mL SEB and 1 µg/mL CD40 for 6 hours, were stained with CD154 antibodies and with a suitable counterstaining. As a control, CD154 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Flow cytometric comparison of different clones for CD154. Human peripheral blood mononuclear cells (PBMCs), stimulated with 1 µg/mL SEB and 1 µg/mL CD40 for 6 hours, were stained with CD154 antibodies and with a suitable counterstaining. As a control, CD154 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Fixation data

To provide an indication on how an antibody performs after fixation of cells, in-house data on staining results before and after fixation with 3.7% formaldehyde using Miltenyi Biotec antibodies are provided. Different experimental settings may lead to different results.

No fixation

Post fixation

Comparison of staining pattern on non-fixed and fixed cells using CD154 (REA238). The performance of the antibody after fixation was tested by comparing the staining pattern on fresh (no fixation) versus fixed (post fixation) cells.

Comparison of staining pattern on non-fixed and fixed cells using CD154 (REA238). The performance of the antibody after fixation was tested by comparing the staining pattern on fresh (no fixation) versus fixed (post fixation) cells.

Specifications for CD154 Antibody, anti-human, REAfinity™

Overview

Clone REA238 recognizes the human CD154 antigen, a 39 kDa transmembrane glycoprotein, also known as CD40L, gp39, T-BAM, TRAP, or Ly-62, which is a proinflammatory and prothrombotic ligand in the tumor necrosis factor family. CD154 is transiently up-regulated on activated CD4

⁺

T cells and plays an important role as a costimulatory molecule in T cell/antigen-presenting cell interactions through ligation of CD40. It mediates B cell proliferation in the absence of co-stimulus as well as IgE production in the presence of IL-4.

Additional information: Clone REA238 displays negligible binding to Fc receptors.

Alternative names

CD40L, CD40-L, gp39, T-BAM, TRAP, Ly-62, HIGM1, IGM

Detailed product information

Technical specifications

Clone	REA238
Clonality	monoclonal
Isotype	recombinant human IgG1
Isotype control	REA Control Antibody (S), human IgG1
Host	human cell line
Type of antibody	Primary antibodies, Recombinant antibodies
Species	human, non-human primate
Cross-reactivity	cynomolgus monkey ( Macaca fascicularis ) , rhesus monkey ( Macaca mulatta )
Antigen	CD154
Alternative names of antigen	CD40L, CD40-L, gp39, T-BAM, TRAP, Ly-62, HIGM1, IGM
Molecular mass of antigen [kDa]	29
Distribution of antigen	dendritic cells, lymphocytes, macrophages, monocytes, T cells, NK cells, mast cells, basophils
Entrez Gene ID	959
RRID	AB_2751146, AB_2751213, AB_2751147, AB_2733341, AB_2733342, AB_2783961, AB_2783960, AB_2751214, AB_2751148, AB_2783959, AB_2783958, AB_2751211, AB_2751145, AB_2801937, AB_2801936, AB_2819450, AB_2819446, AB_2904742, AB_2904741, AB_2751212

Resources for CD154 Antibody, anti-human, REAfinity™

Certificates

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References for CD154 Antibody, anti-human, REAfinity™

Publications

Hollenbaugh, D. et al. (1992) The human T cell antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor: expression of a soluble form of gp39 with B cell co-stimulatory activity. EMBO J. 11(12): 4313-4321
Frentsch, M. et al. (2005)
Direct access to CD4
⁺
T cells specific for defined antigens according to CD154 expression.
Nat Med 11: 1118-1124
Furman, M. et al. (2004) Release of soluble CD40L from platelets is regulated by glycoprotein IIb/IIIa and actin polymerization. J. Am. Coll. Cardiol. 43(12): 2319-2325

Applications

Extended validation