CD47 Antibody, anti-human, REAfinity™

Name: CD47 Antibody, anti-human, REAfinity™
Availability: InStock

CD47 Antibody, anti-human, REAfinity™Extended ValidationRecombinant

Clone:

REA220

Type of antibody:

Primary antibodies, Recombinant antibodies

Isotype:

recombinant human IgG1

Applications:

FC, MICS, IF, IHC, ICC

Alternative names:

Integrin-associated protein, IAP, MER6, OA3, CD47

Flow cytometry applications forCD47 Antibody, anti-human, REAfinity™

APC
APC-Vio 770
Biotin
FITC
PE
PE-Vio 615
PE-Vio 770
Vio Bright B515
Vio Bright R720
VioBlue
VioGreen

Conjugate:

APC

Recommended dilution:

1:50

Remark:

The antibody is suited for staining of formaldehyde-fixed cells.

Tested:

QC tested

Products used:

130-123-315, 130-123-254

Protocols:

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

Description:

Human peripheral blood mononuclear cells (PBMCs) were stained with CD47 antibodies and analyzed by flow cytometry using the MACSQuant^® Analyzer. Lymphocytes were pre-gated for the analysis. The specificity of the conjugated antibodies was confirmed by blocking the binding to the ligand, using pure unconjugated antibodies (left peak). 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 forCD47 Antibody, anti-human, REAfinity™

APC
FITC
PE

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

PFA

Tested:

during development

Species tested:

human

Products used:

130-123-315, 130-123-254

Compatible applications:

IF, IHC, ICC

Protocols:

Protocol for MICS experiments

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

PFA

Tested:

during development

Species tested:

mouse

Products used:

130-103-111

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:

130-123-315, 130-123-254

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

Extended validation for CD47 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 REA220
B6H12	++
2D3	++
CC2C6	++
Cells were incubated with an excess of purified unconjugated CD47 (REA220) 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 CD47 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD47-PE (REA220, red) and counterstained with DRAQ5 (blue) as DNA stain.

Fluorescence microscopy image of CD47 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD47-PE (REA220, red) and counterstained with DRAQ5 (blue) as DNA stain.

Overlay histogram showing flow cytometric analysis of CD47 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD47-PE (REA220). Flow cytometry was performed with the MACSQuant

^®

Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

Overlay histogram showing flow cytometric analysis of CD47 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD47-PE (REA220). Flow cytometry was performed with the MACSQuant

^®

Analyzer. Cell debris and dead cells 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 CD47. Human peripheral blood mononuclear cells (PBMCs) were stained with CD47 antibodies and with a suitable counterstaining. As a control, CD47 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 CD47. Human peripheral blood mononuclear cells (PBMCs) were stained with CD47 antibodies and with a suitable counterstaining. As a control, CD47 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 CD47. Human peripheral blood mononuclear cells (PBMCs) were stained with CD47 antibodies and with a suitable counterstaining. As a control, CD47 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 CD47. Human peripheral blood mononuclear cells (PBMCs) were stained with CD47 antibodies and with a suitable counterstaining. As a control, CD47 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 CD47. Human peripheral blood mononuclear cells (PBMCs) were stained with CD47 antibodies and with a suitable counterstaining. As a control, CD47 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 CD47. Human peripheral blood mononuclear cells (PBMCs) were stained with CD47 antibodies and with a suitable counterstaining. As a control, CD47 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 CD47. Human peripheral blood mononuclear cells (PBMCs) were stained with CD47 antibodies and with a suitable counterstaining. As a control, CD47 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 CD47 (REA220). 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 CD47 (REA220). 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 CD47 (REA220). 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 CD47 (REA220). 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 CD47 Antibody, anti-human, REAfinity™

Overview

Clone REA220 recognizes CD47, a heavily glycosylated protein with an extracellular IgV domain, five transmembrane domains, and a cytoplasmic tail, which exists in four different isoforms. CD47 interact with integrins, thrombospondin-1 (TSP-1), and cell surface gylcoproteins SIRPα and SIRPβ. Owing to these interactions, CD47 plays an important role in lymphocyte homeostasis, maturation and activation of dendritic cells, cellular transmigration, and phagocytosis of cells. Expression of CD47 is found ubiquitously.

Additional information: Clone REA220 displays negligible binding to Fc receptors.

Alternative names

Integrin-associated protein, IAP, MER6, OA3, CD47

Detailed product information

Technical specifications

Clone	REA220
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	CD47
Alternative names of antigen	Integrin-associated protein, IAP, MER6, OA3, CD47
Distribution of antigen	B cells, endothelial cells, epithelial cells, T cells
Entrez Gene ID	961
RRID	AB_2905040, AB_2819555, AB_2819520, AB_2802033, AB_2802017, AB_2905046, AB_2905045, AB_2905039, AB_2905038, AB_2905048, AB_2905047, AB_2658404, AB_2658405, AB_2658412, AB_2658413, AB_2905044, AB_2921867, AB_2921879, AB_2905043, AB_2905042, AB_2905041

Resources for CD47 Antibody, anti-human, REAfinity™

Certificates

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References for CD47 Antibody, anti-human, REAfinity™

Publications

Demeure, C. E. et al. (2000) CD47 engagement inhibits cytokine production and maturation of human dendritic cells. J Immunol 164: 2193-2199
Burger, P. et al. (2012) CD47 functions as a molecular switch for erythrocyte phagocytosis. Blood 119: 5512-5521
Kusakari, S. et al. (2008) Trans-endocytosis of CD47 and SHPS-1 and its role in regulation of the CD47–SHPS-1 system. J. Cell. Sci. 121: 1213-1223

Applications

Extended validation