Clone:
REA611
Type of antibody:
Primary antibodies, Recombinant antibodies
Isotype:
recombinant human IgG1
Applications:
FC, MICS, IF, IHC, MC

Extended validation for CD45RO 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 clonesOverlap in epitope recognition with REA611
UCHL1++
Cells were incubated with an excess of purified unconjugated CD45RO (REA611) 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
View details
Fluorescence microscopy image of CD45RO knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD45RO-PE (REA611, red) and counterstained with DRAQ5 (blue) as DNA stain.
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Fluorescence microscopy image of CD45RO knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD45RO-PE (REA611, red) and counterstained with DRAQ5 (blue) as DNA stain.
Fluorescence microscopy image of CD45RO knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD45RO-PE (REA611, red) and counterstained with DRAQ5 (blue) as DNA stain.
View details
Overlay histogram showing flow cytometric analysis of human CD45RO knockout cells. Wild type (red) and knockout cells (blue) were stained with CD45-PE, clone (REA611). 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 human CD45RO knockout cells. Wild type (red) and knockout cells (blue) were stained with CD45-PE, clone (REA611). 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.
View details
Flow cytometric comparison of different clones for CD45RO. Human peripheral blood mononuclear cells (PBMCs) were stained with CD45RO antibodies and plotted against the side scatter. As a control, CD45RO 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 propidium iodide fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.
View details
Flow cytometric comparison of different clones for CD45RO. Human peripheral blood mononuclear cells (PBMCs) were stained with CD45RO antibodies and plotted against the side scatter. As a control, CD45RO 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 propidium iodide fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.
View details
Flow cytometric comparison of different clones for CD45RO. Human peripheral blood mononuclear cells (PBMCs) were stained with CD45RO antibodies and plotted against the side scatter. As a control, CD45RO 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 propidium iodide fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.
View details
Flow cytometric comparison of different clones for CD45RO. Human peripheral blood mononuclear cells (PBMCs) were stained with CD45RO antibodies and plotted against the side scatter. As a control, CD45RO 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 propidium iodide 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 CD45RO. Human peripheral blood mononuclear cells (PBMCs) were stained with CD45RO antibodies and plotted against the side scatter. As a control, CD45RO 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 propidium iodide 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
View details
Comparison of staining pattern on non-fixed and fixed cells using CD45RO (REA611). The performance of the antibody after fixation was tested by comparing the staining pattern on fresh (no fixation) versus fixed (post fixation) cells.
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Comparison of staining pattern on non-fixed and fixed cells using CD45RO (REA611). 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 CD45RO (REA611). 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 CD45RO Antibody, anti-human, REAfinity™

Overview

Clone REA611 recognizes the human CD45RO antigen, a 180 kDa single-chain membrane glycoprotein, which is an isoform of CD45. CD45RO is expressed by most thymocytes, activated and memory T cells, granulocytes, B cell subsets, and monocytes. CD45RO binds to galectin-1 and CD22. CD45RO antibodies are commonly used in combination with antibodies against CD45RA to discriminate memory T cells from naive T cells.
Additional information: Clone REA611 displays negligible binding to Fc receptors.

Detailed product information

Technical specifications

CloneREA611
Clonalitymonoclonal
Isotyperecombinant human IgG1
Isotype controlREA Control Antibody (S), human IgG1
Hostcell line
Type of antibodyPrimary antibodies, Recombinant antibodies
Specieshuman, non-human primate
Cross-reactivity
chimpanzee (
Pan troglodytes
)
,
common marmoset (
Callithrix jacchus
)
AntigenCD45RO
Molecular mass of antigen [kDa]145
Distribution of antigengranulocytes, T cells, B cells, monocytes, thymocytes
Entrez Gene ID5788
RRIDAB_2733644, AB_2733818, AB_2733819, AB_2733380, AB_2733381, AB_2751196, AB_2751130, AB_2751197, AB_2751131, AB_2751195, AB_2751129, AB_2733129, AB_2733130, AB_2751816, AB_2751760, AB_2811649, AB_2733643

References for CD45RO Antibody, anti-human, REAfinity™

Publications

  1. Charbonneau, H. et al. (1988) The leukocyte common antigen (CD45): a putative receptor-linked protein tyrosine phosphatase. Proc. Natl. Acad. Sci. U.S.A. 85(19): 7182-7186
  2. Trowbridge, I. S. et al. (1994) CD45: an emerging role as a protein tyrosine phosphatase required for lymphocyte activation and development. Annu. Rev. Immunol. 12: 85-116

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