CD45RA Antibody, anti-human, REAfinity™

Name: CD45RA Antibody, anti-human, REAfinity™
Availability: InStock

CD45RA Antibody, anti-human, REAfinity™Extended ValidationRecombinant

Clone:

REA1047

Type of antibody:

Primary antibodies, Recombinant antibodies

Isotype:

recombinant human IgG1

Applications:

FC

Alternative names:

Ptprc, B220, CD45, CD45R, GP180, L-CA, Ly-5, T200

Flow cytometry applications forCD45RA Antibody, anti-human, REAfinity™

APC
APC-Vio 770
Biotin
FITC
PE
PE-Vio 615
PE-Vio 770
PerCP-Vio 700
Vio Bright B515
Vio Bright R720
VioBlue
VioGreen

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:

130-117-853, 130-117-742

Protocols:

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

Description:

Human peripheral blood mononuclear cells (PBMCs) were stained with CD45RA antibodies or with the corresponding REA Control (S) antibodies (left images) as well as with CD45RO antibodies. Flow cytometry was performed 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.

Extended validation for CD45RA 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 REA1047
HI100	++
JS-83	-
L48	-
T6D11	-
REA562	-
Cells were incubated with an excess of purified unconjugated CD45RA (REA1047) 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 CD45RA knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD45RA-PE (REA1047, red) and counterstained with DRAQ5 (blue) as DNA stain.

Fluorescence microscopy image of CD45RA knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD45RA-PE (REA1047, red) and counterstained with DRAQ5 (blue) as DNA stain.

Overlay histogram showing flow cytometric analysis of CD45RA knockout cells. Wild type (red) and knockout cells (blue) were stained with CD45RA-PE, clone (REA1047). 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 CD45RA knockout cells. Wild type (red) and knockout cells (blue) were stained with CD45RA-PE, clone (REA1047). 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 CD45RA. Human peripheral blood mononuclear cells (PBMCs) were stained with CD45RA antibodies and with a suitable counterstaining. As a control, CD45RA 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 CD45RA. Human peripheral blood mononuclear cells (PBMCs) were stained with CD45RA antibodies and with a suitable counterstaining. As a control, CD45RA 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 CD45RA. Human peripheral blood mononuclear cells (PBMCs) were stained with CD45RA antibodies and with a suitable counterstaining. As a control, CD45RA 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 CD45RA. Human peripheral blood mononuclear cells (PBMCs) were stained with CD45RA antibodies and with a suitable counterstaining. As a control, CD45RA 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 CD45RA. Human peripheral blood mononuclear cells (PBMCs) were stained with CD45RA antibodies and with a suitable counterstaining. As a control, CD45RA 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 CD45RA. Human peripheral blood mononuclear cells (PBMCs) were stained with CD45RA antibodies and with a suitable counterstaining. As a control, CD45RA 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 CD45RA (REA1047). 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 CD45RA (REA1047). 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 CD45RA Antibody, anti-human, REAfinity™

Overview

Clone REA1047 recognizes the human CD45RA antigen which is expressed on naive CD4

⁺

and CD8

⁺

T cells as well as on CD8

⁺

effector T cells. CD45RA is also present on subsets of B and NK cells as well as on plasmacytoid dendritic cells. The CD45RA antibody recognizes the 220 kDa isoform of the leukocyte common antigen (LCA), a transmembrane tyrosine phosphatase.

Additional information: Clone REA1047 displays negligible binding to Fc receptors.

Alternative names

Ptprc, B220, CD45, CD45R, GP180, L-CA, Ly-5, T200

Detailed product information

Technical specifications

Clone	REA1047
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, non-human primate
Cross-reactivity	chimpanzee ( Pan troglodytes )
Antigen	CD45RA
Alternative names of antigen	Ptprc, B220, CD45, CD45R, GP180, L-CA, Ly-5, T200
Molecular mass of antigen [kDa]	145
Distribution of antigen	B cells, monocytes, NK cells, T cells, CD4+ T cells, CD8+ T cells, naive T cells, thymocytes
Entrez Gene ID	5788
RRID	AB_2733642, AB_2733814, AB_2733815, AB_2733376, AB_2733377, AB_2733946, AB_2733947, AB_2734090, AB_2734091, AB_2732976, AB_2732977, AB_2733236, AB_2733237, AB_2733125, AB_2733126, AB_2733033, AB_2733034, AB_2734021, AB_2734022, AB_2733524, AB_2733525, AB_2905025, AB_2905024, AB_2733641

Resources for CD45RA Antibody, anti-human, REAfinity™

Certificates

Please follow this

link

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

References for CD45RA Antibody, anti-human, REAfinity™

Publications

Cantisan, S. et al. (2010)
CD45RA expression on HCMV-specific effector memory CD8
⁺
T cells is associated with the duration and intensity of HCMV replication after transplantation.
Clin. Immunol. 137(1): 81-88
Braun, T. et al. (2012)
Effector CD4
⁺
CD45RA
^–
CD25
^bright
Foxp3
^bright
regulatory T cell (eTreg) distribution is significantly impaired in chronic myelomonocytic leukemia (CMML) and correlates with TET 2 mutational status.
Blood 120: 2808-2808

Applications

Extended validation