Clone:
REAL854
Type of antibody:
Releasable antibodies, Primary antibodies, Recombinant antibodies
Applications:
FC
Alternative names:
CEACAM8, CD67, CGM6, NCA-95

Extended validation for CD66b Antibody, anti-human,
REAlease
®

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 REAL854
G10F5+
80H3++
REA306+
Cells were incubated with an excess of purified unconjugated CD66b (REAL854) 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 CD66b. Human peripheral blood mononuclear cells (PBMCs) were first stained with Viobility™ Fixable Dye followed by a suitable counterstaining. Cells were then fixed and permeabilized using the Transcription Factor Staining Buffer Set followed by intracellular staining with CD66b antibodies. As a control, CD66b 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 Viobility 405/520 Fixable Dye 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 CD66b. Human peripheral blood mononuclear cells (PBMCs) were first stained with Viobility™ Fixable Dye followed by a suitable counterstaining. Cells were then fixed and permeabilized using the Transcription Factor Staining Buffer Set followed by intracellular staining with CD66b antibodies. As a control, CD66b 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 Viobility 405/520 Fixable Dye 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 CD66b. Human peripheral blood mononuclear cells (PBMCs) were first stained with Viobility™ Fixable Dye followed by a suitable counterstaining. Cells were then fixed and permeabilized using the Transcription Factor Staining Buffer Set followed by intracellular staining with CD66b antibodies. As a control, CD66b 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 Viobility 405/520 Fixable Dye 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 CD66b. Human peripheral blood mononuclear cells (PBMCs) were first stained with Viobility™ Fixable Dye followed by a suitable counterstaining. Cells were then fixed and permeabilized using the Transcription Factor Staining Buffer Set followed by intracellular staining with CD66b antibodies. As a control, CD66b 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 Viobility 405/520 Fixable Dye 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 CD66b (REAL854). 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 CD66b (REAL854). 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 CD66b (REAL854). 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 CD66b Antibody, anti-human,
REAlease
®

Overview

Clone REAL854 is an antibody fragment derived from the full CD49b antibody molecule. It displays no binding to Fc receptors. The recombinantly engineered antibody fragments are multimerized to form the REAlease Complex to bind markers with high avidity.
Clone REA854 recognizes the human CD66b antigen, a glycosylphosphatidylinositol (GPI) linked protein which is also known as Carcinoembryonic antigen-related cell adhesion molecule 8 (CEACAM8). CD66b is exclusively expressed on neutrophils and eosinophils and on leukocytes of chronic myeloid Leukemia patients and bone marrow. Under normal conditions, neutrophils have minimal expression of CD66b, but incubation in vitro with inflammatory agonists rapidly increases this expression. Furthermore, expression of CD66b in eosinophils is enhanced in vivo in patients with a helminth infection, specifically with Schistosoma mansoni. The eosinophil expression of CD66b is also enhanced in vitro by incubation with activated lymphocytes or by brief incubation with IL-5. Crosslinking of CD66b on peripheral blood neutrophils mediates the release of IL-8 from intracellular storage.
The REAlease Kits consist of the respective fluorochrome-conjugated REAlease Complexes and the REAlease Support Kit for removal of the REAlease Complexes and optional relabeling with different fluorochrome-conjugated REAlease Complexes.

Alternative names

CEACAM8, CD67, CGM6, NCA-95

Detailed product information

Technical specifications

CloneREAL854
Clonalitymonoclonal
Isotype controlControl Antibody
Hostcell line
Type of antibodyReleasable antibodies, Primary antibodies, Recombinant antibodies
Specieshuman
AntigenCD66b
Alternative names of antigenCEACAM8, CD67, CGM6, NCA-95
Distribution of antigengranulocytes, eosinophils, neutrophils, bone marrow

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