CD119 Antibody, anti-human, REAfinity™

Name: CD119 Antibody, anti-human, REAfinity™
Availability: InStock

CD119 Antibody, anti-human, REAfinity™Extended ValidationRecombinant

Clone:

REA161

Type of antibody:

Recombinant antibodies, Primary antibodies

Isotype:

recombinant human IgG1

Applications:

FC

Alternative names:

IFNGR1, Ifngr, IFN-γRα

Flow cytometry applications forCD119 Antibody, anti-human, REAfinity™

APC
APC-Vio 770
Biotin
FITC
PE
PE-Vio 615
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

Products used:

130-125-869, 130-125-846

Protocols:

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

Description:

Human peripheral blood mononuclear cells (PBMCs) were stained with CD119 antibodies and analyzed by flow cytometry using the MACSQuant^® Analyzer. The specificity of the conjugated antibodies was confirmed by blocking the binding to the ligand, using pure unconjugated antibodies (left peak). Monocytes were pre-gated for the analysis. 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.

Extended validation for CD119 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 REA161
GIR-208	-
GIR-94	++
Cells were incubated with an excess of purified unconjugated CD119 (REA161) 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 CD119 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD119-PE (REA161, red) and counterstained with DRAQ5 (blue) as DNA stain.

Fluorescence microscopy image of CD119 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD119-PE (REA161, red) and counterstained with DRAQ5 (blue) as DNA stain.

Overlay histogram showing flow cytometric analysis of CD119 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD119-PE, clone (REA161). 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 CD119 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD119-PE, clone (REA161). 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 CD119. Human peripheral blood mononuclear cells (PBMCs) were stained with CD119 antibodies and with a suitable counterstaining. As a control, CD119 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 CD119. Human peripheral blood mononuclear cells (PBMCs) were stained with CD119 antibodies and with a suitable counterstaining. As a control, CD119 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 CD119. Human peripheral blood mononuclear cells (PBMCs) were stained with CD119 antibodies and with a suitable counterstaining. As a control, CD119 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 CD119. Human peripheral blood mononuclear cells (PBMCs) were stained with CD119 antibodies and with a suitable counterstaining. As a control, CD119 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 CD119 (REA161). 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 CD119 (REA161). 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 CD119 Antibody, anti-human, REAfinity™

Overview

Clone REA161 recognizes CD119, the alpha chain subunit of the IFN-γ receptor. CD119 is a 80–100 kDa type I transmembrane protein and is responsible for IFN-γ binding. Along with the β chain, CD119 is also involved in signal transduction via STAT1 activation. Except for mature erythrocytes, expression of CD119 is found on the surface of most human cells.

Additional information: Clone REA161 displays negligible binding to Fc receptors.

Alternative names

IFNGR1, Ifngr, IFN-γRα

Detailed product information

Technical specifications

Clone	REA161
Clonality	monoclonal
Isotype	recombinant human IgG1
Isotype control	REA Control Antibody (S), human IgG1
Host	human cell line
Type of antibody	Recombinant antibodies, Primary antibodies
Species	human
Antigen	CD119
Alternative names of antigen	IFNGR1, Ifngr, IFN-γRα
Molecular mass of antigen [kDa]	53
Distribution of antigen	B cells, T cells, dendritic cells, endothelial cells, macrophages
Entrez Gene ID	3459
RRID	AB_2922089, AB_2922056, AB_2857805, AB_2857812, AB_2857802, AB_2654598, AB_2654599, AB_2654604, AB_2654605, AB_2654606, AB_2654607, AB_2654608, AB_2654609, AB_2857815

Resources for CD119 Antibody, anti-human, REAfinity™

Certificates

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References for CD119 Antibody, anti-human, REAfinity™

Publications

Harris, D. P. et al. (2005) Regulation of IFN-γ production by B effector 1 cells: Essential roles for T-bet and the IFN-γ receptor. J Immunol 174: 6781 -6790
Basler, C. F. et al. (2002) Viruses and the type I interferon antiviral system: induction and evasion. Int. Rev. Immunol. 21(4-5): 305-337

Applications

Extended validation