CD312 (EMR2) Antibody, anti-human, REAfinity™

Name: CD312 (EMR2) Antibody, anti-human, REAfinity™
Availability: InStock

CD312 (EMR2) Antibody, anti-human, REAfinity™Extended ValidationRecombinant

Clone:

REA302

Type of antibody:

Primary antibodies, Recombinant antibodies

Isotype:

recombinant human IgG1

Applications:

FC, MICS, IF, IHC

Alternative names:

ADGRE2, EMR2

Flow cytometry applications forCD312 (EMR2) Antibody, anti-human, REAfinity™

APC
APC-Vio 770
Biotin
FITC
PE
PE-Vio 770

Conjugate:

APC

Recommended dilution:

1:50

Remark:

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

Tested:

QC tested

Reported:

29017060,

Products used:

130-124-452, 130-124-443

Protocols:

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

Description:

Human peripheral blood cells after erythrocyte lysis were stained with CD312 (EMR2) antibodies or with the corresponding REA Control (S) antibodies (left images) as well as with CD14 antibodies and 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 or 4',6-diamidino-2-phenylindole (DAPI) fluorescence, as in the case of tandem conjugates.

MICS (MACSima Imaging Cyclic Staining) applications forCD312 (EMR2) Antibody, anti-human, REAfinity™

APC
FITC
PE

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

Acetone

Tested:

during development

Species tested:

human

Products used:

130-124-452, 130-124-443

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

Extended validation for CD312 (EMR2) 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 REA302
2A1	++
W15101A	++
Cells were incubated with an excess of purified unconjugated CD312 (EMR2) (REA302) 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.

Knockout validation

To ensure antibody specificity, the target gene is knocked out in a suitable cell line using the CRISPR/Cas9 system and the knockout is confirmed by sequencing of the target locus. The antibody is considered to bind specifically to the intended epitope if no antibody binding to the knockout cells can be detected. The antibody staining is controlled by fluorescence microscopy and/or flow cytometry.

WT

KO

Fluorescence microscopy image of CD312 (EMR2) knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD312 (EMR2)-PE (REA302, red) and counterstained with DRAQ5 (blue) as DNA stain.

Fluorescence microscopy image of CD312 (EMR2) knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD312 (EMR2)-PE (REA302, red) and counterstained with DRAQ5 (blue) as DNA stain.

Overlay histogram showing flow cytometric analysis of CD312 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD312-PE, clone (REA302). 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 propidium iodide fluorescence.

Overlay histogram showing flow cytometric analysis of CD312 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD312-PE, clone (REA302). 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 propidium iodide fluorescence.

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 CD312 (EMR2). Human peripheral blood mononuclear cells (PBMCs) after erythrocyte lysis were stained with CD312 (EMR2) antibodies and with a suitable counterstaining. As a control, CD312 (EMR2) 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 CD312 (EMR2). Human peripheral blood mononuclear cells (PBMCs) after erythrocyte lysis were stained with CD312 (EMR2) antibodies and with a suitable counterstaining. As a control, CD312 (EMR2) 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 CD312 (EMR2). Human peripheral blood mononuclear cells (PBMCs) after erythrocyte lysis were stained with CD312 (EMR2) antibodies and with a suitable counterstaining. As a control, CD312 (EMR2) 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 CD312 (EMR2) (REA302). 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 CD312 (EMR2) (REA302). 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 CD312 (EMR2) Antibody, anti-human, REAfinity™

Overview

Clone REA302 recognizes the human CD312 antigen, a multi-pass membrane protein also known as EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2) which shares strikingly similar molecular characteristics with CD97. CD312 is a member of the epidermal growth factor-seven transmembrane (EGF-TM7) family of proteins expressed predominantly by cells of the immune system. CD312 fails to interact with CD55, the cellular ligand for CD97, suggesting the possibility of different cellular ligands. CD312 interacts with chondroitin sulphate glycosaminoglycans in an isoform-specific manner. Expression of CD312 is up-regulated during differentiation and maturation of macrophages, and is conversely down-regulated during dendritic cell maturation. In monocytes and macrophages, CD312 can be specifically up-regulated by LPS and interleukin 10 (IL-10) via an IL-10–mediated pathway. In inflamed tissues, CD312 is detected in subpopulations of myeloid cells including macrophages and neutrophils. CD312 plays a role in the migration and adhesion of myeloid cells during cell differentiation, maturation, and activation.

Additional information: Clone REA302 displays negligible binding to Fc receptors.

Alternative names

ADGRE2, EMR2

Detailed product information

Technical specifications

Clone	REA302
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	CD312 (EMR2)
Alternative names of antigen	ADGRE2, EMR2
Molecular mass of antigen [kDa]	88
Distribution of antigen	myeloid leukemia cells, granulocytes, neutrophils
Entrez Gene ID	30817
RRID	AB_2751831, AB_2819623, AB_2819619, AB_2657349, AB_2657350, AB_2657351, AB_2657352, AB_2657353, AB_2657354, AB_2904934, AB_2904933, AB_2904932, AB_2904931, AB_2751862

Resources for CD312 (EMR2) Antibody, anti-human, REAfinity™

Certificates

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References for CD312 (EMR2) Antibody, anti-human, REAfinity™

Publications

Kwakkenbos, M. J. et al. (2002) The human EGF-TM7 family member EMR2 is a heterodimeric receptor expressed on myeloid cells. J. Leukoc. Biol. 71(5): 854-862
Chang, G. W. et al. (2007) CD312, the human adhesion-GPCR EMR2, is differentially expressed during differentiation, maturation, and activation of myeloid cells. Biochem. Biophys. Res. Commun. 353(1): 133-138
Yona, S. et al. (2008) Ligation of the adhesion-GPCR EMR2 regulates human neutrophil function. FASEB J. 22(3): 741-751

Applications

Extended validation