Clone:
5C8
Type of antibody:
Primary antibodies
Isotype:
mouse IgG2aκ
Applications:
ICFC
Alternative names:
CD40LG, CD40L, HIGM1, IGM, IMD3, T-BAM, TNFSF5, TRAP, gp39

Extended validation for CD154 Antibody, anti-human

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 clonesOverlap in epitope recognition with 5C8
24-31++
REA238++
TRAP1+
Cells were incubated with an excess of purified unconjugated CD154 (5C8) 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.
View details
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.
View details
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.
View details
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.
View details
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.
View details
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
View details
Comparison of staining pattern on non-fixed and fixed cells using CD154 (5C8). The performance of the antibody after fixation was tested by comparing the staining pattern on fresh (no fixation) versus fixed (post fixation) cells.
View details
Comparison of staining pattern on non-fixed and fixed cells using CD154 (5C8). 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 (5C8). 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

Overview

The antibody specifically recognizes the human CD154 antigen, a 39 kDa transmembrane glycoprotein, also known as CD40L, gp39, T-BAM, TRAP, or Ly-62. 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. The antibody has been shown to block the activation of antigen-presenting cells by T helper cells
in vitro
. Because of its transient expression within hours after activation, CD154 can be used as a marker for activated antigen-specific CD4
+
T cells. The addition of a CD40-blocking antibody during the stimulation of cell suspensions prevents the down-regulation of CD154 expression induced by interaction with CD40 expressed on antigen-presenting cells. For intracellular detection of CD154 expression or if a pure population of enriched T cells is used, blocking of CD40 is not required.

Alternative names

CD40LG, CD40L, HIGM1, IGM, IMD3, T-BAM, TNFSF5, TRAP, gp39

Detailed product information

Technical specifications

Clone5C8
Clonalitymonoclonal
Isotypemouse IgG2aκ
Isotype controlIsotype Control Antibody, mouse IgG2a
Hostmouse
Type of antibodyPrimary antibodies
Specieshuman, non-human primate
Cross-reactivity
rhesus monkey (
Macaca mulatta
)
,
cynomolgus monkey (
Macaca fascicularis
)
AntigenCD154
Alternative names of antigenCD40LG, CD40L, HIGM1, IGM, IMD3, T-BAM, TNFSF5, TRAP, gp39
Molecular mass of antigen [kDa]29
Distribution of antigendendritic cells, lymphocytes, macrophages, monocytes, T cells, NK cells, mast cells, basophils
Entrez Gene ID959
RRIDAB_2751141, AB_2751208, AB_2751142, AB_2726468, AB_2726191, AB_2751210, AB_2751144, AB_2751209, AB_2751143, AB_2751205, AB_2751139, AB_2751206, AB_2751140, AB_2751207

References for CD154 Antibody, anti-human

Publications

  1. Frentsch, M. et al. (2005)
    Direct access to CD4
    +
    T cells specific for defined antigens according to CD154 expression.
    Nat Med 11: 1118-1124

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