CD45 Antibody, anti-non-human primate, REAfinity™

Name: CD45 Antibody, anti-non-human primate, REAfinity™
Availability: InStock

CD45 Antibody, anti-non-human primate, REAfinity™Extended ValidationRecombinant

Clone:

REA1023

Type of antibody:

Primary antibodies, Recombinant antibodies

Isotype:

recombinant human IgG1

Applications:

FC, MICS, IF, IHC

Alternative names:

LCA, L-CA, T200

Flow cytometry applications forCD45 Antibody, anti-non-human primate, REAfinity™

APC
Biotin
FITC
PE
PE-Vio 615
PE-Vio 770
PerCP-Vio 700
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:

130-117-282, 130-117-191

Protocols:

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

Description:

Peripheral blood mononuclear cells (PBMCs) from rhesus monkey were stained with CD45 antibodies or with the corresponding REA Control (S) antibodies (left images) 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 fluorescence or 4',6-diamidino-2-phenylindole (DAPI) fluorescence, as in the case of tandem conjugates.

MICS (MACSima Imaging Cyclic Staining) applications forCD45 Antibody, anti-non-human primate, REAfinity™

APC
FITC
PE

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

PFA

Tested:

during development

Species tested:

human

Products used:

130-117-282, 130-117-191

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:

130-117-282, 130-117-191

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

Extended validation for CD45 Antibody, anti-non-human primate, 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 REA1023
5B1	-
REA747	-
2D1	-
HI30	-
REAL258	-
MB4-6D6	++
Cells were incubated with an excess of purified unconjugated CD45 (REA1023) 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 CD45 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD45-PE (REA1023, red) and counterstained with DRAQ5 (blue) as DNA stain.

Fluorescence microscopy image of CD45 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD45-PE (REA1023, red) and counterstained with DRAQ5 (blue) as DNA stain.

Overlay histogram showing flow cytometric analysis of human CD45 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD45-PE, clone (REA1023). 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 CD45 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD45-PE, clone (REA1023). 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 CD45. Rhesus monkey peripheral blood mononuclear cells (PBMCs) were stained with CD45 antibodies and plotted against the side scatter. As a control, CD45 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 CD45. Rhesus monkey peripheral blood mononuclear cells (PBMCs) were stained with CD45 antibodies and plotted against the side scatter. As a control, CD45 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 CD45. Rhesus monkey peripheral blood mononuclear cells (PBMCs) were stained with CD45 antibodies and plotted against the side scatter. As a control, CD45 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 CD45 (REA1023). 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 CD45 (REA1023). 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 CD45 Antibody, anti-non-human primate, REAfinity™

Overview

Clone REA1023 recognizes the rhesus monkey (

Macaca mulatta

) and cynomolgus monkey (

Macaca fascicularis

) CD45 antigen. It is reported to cross-react with pigtail monkey (

Macaca nemestrina

) and human. CD45 is a type I membrane glycoprotein also known as leukocyte common antigen (LCA), T200, or receptor-type tyrosine-protein phosphatase C (PTPRC). It is expressed on non-human primate hematopietic cells, such as lymphocytes, monocytes, and granulocytes. It is not found on erythroid cells. CD45 functions as a regulator for cell growth, differentiation, cell cycle, and oncogenic transformation.

Additional information: Clone REA1023 displays negligible binding to Fc receptors.

Alternative names

LCA, L-CA, T200

Detailed product information

Technical specifications

Clone	REA1023
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	non-human primate, human
Cross-reactivity	rhesus monkey ( Macaca mulatta ) , cynomolgus monkey ( Macaca fascicularis ) , human, pigtail monkey ( Macaca nemestrina )
Antigen	CD45
Alternative names of antigen	LCA, L-CA, T200
Molecular mass of antigen [kDa]	41
Distribution of antigen	granulocytes, lymphocytes, monocytes, leukocytes
Entrez Gene ID	5788
RRID	AB_2727862, AB_2727900, AB_2727863, AB_2727901, AB_2727864, AB_2727902, AB_2727865, AB_2727903, AB_2727866, AB_2727904, AB_2727867, AB_2727905, AB_2727868, AB_2727869, AB_2727906, AB_2727870, AB_2921928, AB_2921902, AB_2727899

Resources for CD45 Antibody, anti-non-human primate, REAfinity™

Certificates

Please follow this

link

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

Applications

Extended validation