CD66b Antibody, anti-human,
REAlease
^®

Name: CD66b Antibody, anti-human, REAlease®
Availability: InStock

CD66b Antibody, anti-human,
REAlease
^®
Extended ValidationRecombinant

Clone:

REAL854

Type of antibody:

Releasable antibodies, Primary antibodies, Recombinant antibodies

Applications:

FC

Alternative names:

CEACAM8, CD67, CGM6, NCA-95

Flow cytometry applications forCD66b Antibody, anti-human,
REAlease
^®

FITC

Conjugate:

FITC

Recommended dilution:

1:50

Tested:

QC tested

Products used:

130-127-332, 130-127-314

Protocols:

Cell surface staining protocol using REAlease™ antibodies

Description:

Human peripheral blood cells after erythrocyte lysis were stained with REAlease CD66b-FITC (B) or with the corresponding REAL Control (A). REAlease CD66b-FITC was released using the REAlease Release Reagent (C). To show epitope accessibility after REAlease Complex release, the cells were washed with the REAlease Stop Reagent and restained with REAlease CD66b-FITC (D). Cells were analyzed by flow cytometry using the MACSQuant^® Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide (PI) fluorescence.

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 clones	Overlap 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.

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.

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

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

Clone	REAL854
Clonality	monoclonal
Isotype control	Control Antibody
Host	cell line
Type of antibody	Releasable antibodies, Primary antibodies, Recombinant antibodies
Species	human
Antigen	CD66b
Alternative names of antigen	CEACAM8, CD67, CGM6, NCA-95
Distribution of antigen	granulocytes, eosinophils, neutrophils, bone marrow
RRID	AB_2905102, AB_2905103

Resources for CD66b Antibody, anti-human,
REAlease
^®

Certificates

Please follow this

link

to search for Certificates of Analysis (CoA) by lot number.

Applications

Extended validation