CD3 Antibody, anti-human, REAfinity™

Name: CD3 Antibody, anti-human, REAfinity™
Availability: InStock

CD3 Antibody, anti-human, REAfinity™Extended ValidationRecombinant

Clone:

REA613

Type of antibody:

Primary antibodies, Recombinant antibodies

Isotype:

recombinant human IgG1

Applications:

FC, MICS, IF, IHC, MC, 3D-IF

Alternative names:

CD3-epsilon, CD3e, Leu4, T-cell surface antigen T3/Leu-4 epsilon chain, T3E, IMD18, TCRE

Flow cytometry applications forCD3 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

Reported:

31171652,

Products used:

130-113-697, 130-113-135

Protocols:

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

Description:

Human peripheral blood mononuclear cells (PBMCs) were stained with CD3 antibodies or with the corresponding REA Control (S) antibodies (left images) as well as with CD4 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.

MICS (MACSima Imaging Cyclic Staining) applications forCD3 Antibody, anti-human, REAfinity™

APC
FITC
PE

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

PFA

Tested:

during development

Species tested:

human

Products used:

130-113-697, 130-113-135

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:

130-113-697, 130-113-135

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

3D-Immunofluorescence applications forCD3 Antibody, anti-human, REAfinity™

Vio R667

Conjugate:

Vio R667

Recommended dilution:

1:50

Tested:

during development

Species tested:

human

Products used:

130-128-740

Tissue clearing:

MACS^® Clearing Kit

Compatible applications:

IF, IHC

Protocols:

Immunostaining and clearing of primary human tumors and patient-derived xenografts for 3D imaging ...

Description:

3D-immunofluorescence analysis of a human tumor xenograft fixed with 4% PFA and subjected to whole-mount immunostaining with CD3 Antibody, anti-human, Vio R667 (red). The sample was processed and optically cleared using the MACS^® Clearing Kit and imaged with an UltraMicroscope II light sheet instrument. 3D-rendered maximum intensity projection of sample zoom-in (upper left, Rhodamine-lectin in grey), zoom-out (upper right), overview (lower left, autofluorescenec in grey) and video (lower right, Rhodamine Lectin in grey). Scale bars: 300 µm (upper left), 1000 µm (upper right, lower left).

Mass cytometry applications forCD3 Antibody, anti-human, REAfinity™

pure

Conjugate:

pure

Reported:

32676612,

Products used:

130-122-282

Extended validation for CD3 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 recognition with REA613
BW264/56	++
UCHT1	++
HIT3	++
SK7	++
OKT3	++
SP34-2	+
Cells were incubated with an excess of purified unconjugated CD3 (REA613) 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 CD3 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD3-PE (REA613, red) and counterstained with DRAQ5 (blue) as DNA stain.

Fluorescence microscopy image of CD3 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD3-PE (REA613, red) and counterstained with DRAQ5 (blue) as DNA stain.

Overlay histogram showing flow cytometric analysis of human CD3 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD3-PE, clone (REA613). 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 CD3 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD3-PE, clone (REA613). 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 CD3. Human peripheral blood mononuclear cells (PBMCs) were stained with CD3 antibodies and with a suitable counterstaining. As a control, CD3 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 CD3. Human peripheral blood mononuclear cells (PBMCs) were stained with CD3 antibodies and with a suitable counterstaining. As a control, CD3 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 CD3. Human peripheral blood mononuclear cells (PBMCs) were stained with CD3 antibodies and with a suitable counterstaining. As a control, CD3 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 CD3. Human peripheral blood mononuclear cells (PBMCs) were stained with CD3 antibodies and with a suitable counterstaining. As a control, CD3 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 CD3. Human peripheral blood mononuclear cells (PBMCs) were stained with CD3 antibodies and with a suitable counterstaining. As a control, CD3 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 CD3. Human peripheral blood mononuclear cells (PBMCs) were stained with CD3 antibodies and with a suitable counterstaining. As a control, CD3 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 CD3. Human peripheral blood mononuclear cells (PBMCs) were stained with CD3 antibodies and with a suitable counterstaining. As a control, CD3 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 CD3. Human peripheral blood mononuclear cells (PBMCs) were stained with CD3 antibodies and with a suitable counterstaining. As a control, CD3 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 CD3. Human peripheral blood mononuclear cells (PBMCs) were stained with CD3 antibodies and with a suitable counterstaining. As a control, CD3 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 CD3 (REA613). 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 CD3 (REA613). 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 CD3 Antibody, anti-human, REAfinity™

Overview

Clone REA613 recognizes the human CD3 antigen, a single-pass type I membrane protein, also known as Leu4. CD3 is present on mature human T cells, thymocytes, a subset of natural killer (NK) cells, and on Purkinje cells in cerebellum. It is associated with the T cell receptor (TCR) and is responsible for its signal transduction. The CD3 antigen is a complex of five invariable chains: γ, δ, ε, ζ, and η. The epitope recognized by the antibody is located on the ε-chain of the CD3 complex.

Additional information: Clone REA613 displays negligible binding to Fc receptors.

Alternative names

CD3-epsilon, CD3e, Leu4, T-cell surface antigen T3/Leu-4 epsilon chain, T3E, IMD18, TCRE

Detailed product information

Technical specifications

Clone	REA613
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	CD3
Alternative names of antigen	CD3-epsilon, CD3e, Leu4, T-cell surface antigen T3/Leu-4 epsilon chain, T3E, IMD18, TCRE
Molecular mass of antigen [kDa]	21
Distribution of antigen	leukocytes, NK cells, T cells, thymocytes, CD4+ T cells, CD8+ T cells, NKT cells, T helper cells
Entrez Gene ID	916
RRID	AB_2725966, AB_2726242, AB_2725967, AB_2726238, AB_2725963, AB_2726771, AB_2726687, AB_2726245, AB_2725970, AB_2726772, AB_2726688, AB_2726243, AB_2725968, AB_2726239, AB_2725964, AB_2726244, AB_2725969, AB_2726240, AB_2725965, AB_2801863, AB_2889574, AB_2889573, AB_2904991, AB_2904990, AB_2904992, AB_2726241

Resources for CD3 Antibody, anti-human, REAfinity™

Documents and Protocols

Scientific posters

Multicolor immunophenotyping of human tumor cells using MACSQuant® Analyzer 16

Scientific posters

Complete workflows allow comprehensive tumor microenvironment analysis of cell subsets from limited tumor patient samples with the MACSima™ Imaging Platform

App notes/customer reports

Flow cytometry analysis of whole-blood NK cells expressing single killer cell immunoglobulin–like receptors

App notes/customer reports

Comprehensive flow cytometry Analysis of isolated NK cells from whole blood.

Certificates

Please follow this

link

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

References for CD3 Antibody, anti-human, REAfinity™

Publications

Gold, D. P. et al. (1986) Isolation of cDNA clones encoding the 20K non-glycosylated polypeptide chain of the human T-cell receptor/T3 complex. Nature 321(6068): 431-434
Birnbaum, M. E. et al. (2014) Molecular architecture of the αβ T cell receptor-CD3 complex. Proc. Natl. Acad. Sci. U.S.A. 111(49): 17576-17581
D'Acquisto, F. et al. (2011)
CD3
⁺
CD4
^-
CD8
^-
(double negative) T cells: saviours or villains of the immune response?
Biochem. Pharmacol. 82(4): 333-340

Applications

Extended validation