CD66b Antibody, anti-human, REAfinity™

Name: CD66b Antibody, anti-human, REAfinity™
Availability: InStock

CD66b Antibody, anti-human, REAfinity™Extended ValidationRecombinant

Clone:

REA306

Type of antibody:

Primary antibodies, Recombinant antibodies

Isotype:

recombinant human IgG1

Applications:

FC, MICS, IF, IHC, MC

Alternative names:

CEACAM8, CD67, CGM6, NCA-95

Flow cytometry applications forCD66b Antibody, anti-human, REAfinity™

APC
APC-Vio 770
Biotin
FITC
PE
PE-Vio 770
PerCP-Vio 700
Vio Bright B515
Vio Bright V423

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:

130-117-804, 130-117-692

Protocols:

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

Description:

Human peripheral blood cells after erythrocyte lysis were stained with CD66b antibodies and 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.

MICS (MACSima Imaging Cyclic Staining) applications forCD66b Antibody, anti-human, REAfinity™

APC
FITC
PE

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

FFPE

Tested:

during development

Species tested:

human

Products used:

130-117-804, 130-117-692

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

Acetone

Tested:

during development

Species tested:

human

Products used:

130-117-804, 130-117-692

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

Mass cytometry applications forCD66b Antibody, anti-human, REAfinity™

pure

Conjugate:

pure

Reported:

32676612,

Products used:

130-108-019

Extended validation for CD66b 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 REA306
G10F5	++
80H3	-
Cells were incubated with an excess of purified unconjugated CD66b (REA306) 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) after erythrocyte lysis were stained with CD66b antibodies and plotted against the side scatter. 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 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 CD66b. Human peripheral blood mononuclear cells (PBMCs) after erythrocyte lysis were stained with CD66b antibodies and plotted against the side scatter. 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 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 CD66b. Human peripheral blood mononuclear cells (PBMCs) after erythrocyte lysis were stained with CD66b antibodies and plotted against the side scatter. 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 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 CD66b. Human peripheral blood mononuclear cells (PBMCs) after erythrocyte lysis were stained with CD66b antibodies and plotted against the side scatter. 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 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 CD66b. Human peripheral blood mononuclear cells (PBMCs) after erythrocyte lysis were stained with CD66b antibodies and plotted against the side scatter. 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 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 CD66b (REA306). 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 (REA306). 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, REAfinity™

Overview

Clone REA306 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 interleukin 5 (IL-5). Crosslinking of CD66b on peripheral blood neutrophils mediates the release of IL-8 from intracellular storage.

Additional information: Clone REA306 displays negligible binding to Fc receptors.

Alternative names

CEACAM8, CD67, CGM6, NCA-95

Detailed product information

Technical specifications

Clone	REA306
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
Antigen	CD66b
Alternative names of antigen	CEACAM8, CD67, CGM6, NCA-95
Molecular mass of antigen [kDa]	32
Distribution of antigen	granulocytes, eosinophils, neutrophils, bone marrow
Entrez Gene ID	1088
RRID	AB_2733400, AB_2733532, AB_2733533, AB_2751860, AB_2751829, AB_2752011, AB_2751981, AB_2811418, AB_2811406, AB_2802064, AB_2802056, AB_2658994, AB_2905101, AB_2905100, AB_2905099, AB_2905098, AB_2733399

Resources for CD66b Antibody, anti-human, REAfinity™

Documents and Protocols

Brochures/posters

REAfinity™ Antibodies for mass cytometry - Recombinantly engineered to increase reproducibility

Certificates

Please follow this

link

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

References for CD66b Antibody, anti-human, REAfinity™

Publications

Yoon, J. et al. (2007) CD66b regulates adhesion and activation of human eosinophils. J Immunol 179(12): 8454-8462
Molgora, M. et al. (2017) IL-1R8 is a checkpoint in NK cells regulating anti-tumour and anti-viral activity. Nature 551(7678): 110-114
Eades-Perner, A. M. et al. (1998) Mice transgenic for the human CGM6 gene express its product, the granulocyte marker CD66b, exclusively in granulocytes. Blood 91(2): 663-672
Berling, B. et al. (1990) Cloning of a carcinoembryonic antigen gene family member expressed in leukocytes of chronic myeloid leukemia patients and bone marrow. Cancer Res. 50(20): 6534-6539
Pearl, M. L. et al. (2014) Prognostic analysis of invasive circulating tumor cells (iCTCs) in epithelial ovarian cancer. Gynecol. Oncol. 134(3): 581-590
Liakopoulos, V. et al. (2018) Hemodialysis-related changes in phenotypical features of monocytes. Sci Rep 8(1): 13964

Applications

Extended validation