CD56 Antibody, anti-human, REAfinity™

Name: CD56 Antibody, anti-human, REAfinity™
Availability: InStock

CD56 Antibody, anti-human, REAfinity™Extended ValidationRecombinant

Clone:

REA196

Type of antibody:

Primary antibodies, Recombinant antibodies

Isotype:

recombinant human IgG1

Applications:

FC, MC

Alternative names:

NCAM1, MSK39, NCAM

Flow cytometry applications forCD56 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 R667
Vio Bright R720
Vio Bright V423

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:

130-113-872, 130-113-310

Protocols:

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

Description:

Human peripheral blood mononuclear cells (PBMCs) were stained with CD56 antibodies or with the corresponding REA Control (S) antibodies (left images) as well as with CD3 antibodies. Cells were then analyzed by flow cytometry 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.

Mass cytometry applications forCD56 Antibody, anti-human, REAfinity™

pure

Conjugate:

pure

Reported:

32676612,

Products used:

130-108-016

Extended validation for CD56 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 REA196
NCAM16.2	++
TULY56	++
5.1H11	++
AF12-7H3	-
C5.9	++
N901/NKH1	++
CMSSB	+
B159	-
HCD56	-
MEM188	-
Cells were incubated with an excess of purified unconjugated CD56 (REA196) 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 CD56 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD56-PE (REA196, red) and counterstained with DRAQ5 (blue) as DNA stain.

Fluorescence microscopy image of CD56 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD56-PE (REA196, red) and counterstained with DRAQ5 (blue) as DNA stain.

Overlay histogram showing flow cytometric analysis of CD56 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD56-PE, clone (REA196). 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 CD56 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD56-PE, clone (REA196). 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 CD56. Human peripheral blood mononuclear cells (PBMCs) were stained with CD56 antibodies and with a suitable counterstaining. As a control, CD56 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 CD56. Human peripheral blood mononuclear cells (PBMCs) were stained with CD56 antibodies and with a suitable counterstaining. As a control, CD56 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 CD56. Human peripheral blood mononuclear cells (PBMCs) were stained with CD56 antibodies and with a suitable counterstaining. As a control, CD56 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 CD56. Human peripheral blood mononuclear cells (PBMCs) were stained with CD56 antibodies and with a suitable counterstaining. As a control, CD56 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 CD56. Human peripheral blood mononuclear cells (PBMCs) were stained with CD56 antibodies and with a suitable counterstaining. As a control, CD56 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 CD56. Human peripheral blood mononuclear cells (PBMCs) were stained with CD56 antibodies and with a suitable counterstaining. As a control, CD56 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 CD56. Human peripheral blood mononuclear cells (PBMCs) were stained with CD56 antibodies and with a suitable counterstaining. As a control, CD56 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 CD56. Human peripheral blood mononuclear cells (PBMCs) were stained with CD56 antibodies and with a suitable counterstaining. As a control, CD56 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 CD56. Human peripheral blood mononuclear cells (PBMCs) were stained with CD56 antibodies and with a suitable counterstaining. As a control, CD56 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 CD56. Human peripheral blood mononuclear cells (PBMCs) were stained with CD56 antibodies and with a suitable counterstaining. As a control, CD56 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 CD56. Human peripheral blood mononuclear cells (PBMCs) were stained with CD56 antibodies and with a suitable counterstaining. As a control, CD56 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 CD56 (REA196). 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 CD56 (REA196). 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 CD56 Antibody, anti-human, REAfinity™

Overview

Clone REA196 recognizes the human CD56 antigen, a glycoprotein of the Ig-superfamily, also known as neural cell adhesion molecule (NCAM), which is expressed in blood on practically all resting and activated NK cells and on a minor subset of CD3

⁺

T cells. CD56 is also expressed in brain (cerebellum and cortex) and at neuromuscular junctions. Certain large granular lymphocyte (LGL) leukemias, small-cell lung carcinomas, neuronal-derived tumors, myelomas, and myeloid leukemias also express CD56.

Additional information: Clone REA196 displays negligible binding to Fc receptors.

Alternative names

NCAM1, MSK39, NCAM

Detailed product information

Technical specifications

Clone	REA196
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 ) , rhesus monkey ( Macaca mulatta ) , cynomolgus monkey ( Macaca fascicularis ) , olive baboon ( Papio anubis )
Antigen	CD56
Alternative names of antigen	NCAM1, MSK39, NCAM
Molecular mass of antigen [kDa]	92
Distribution of antigen	NK cells, T cells
Entrez Gene ID	4684
RRID	AB_2726695, AB_2726367, AB_2726090, AB_2726365, AB_2726088, AB_2726780, AB_2726696, AB_2726368, AB_2726091, AB_2733135, AB_2733136, AB_2733037, AB_2733038, AB_2726781, AB_2726697, AB_2726366, AB_2726089, AB_2819377, AB_2819374, AB_2658728, AB_2905076, AB_2905077, AB_2905075, AB_2905074, AB_2726779

Resources for CD56 Antibody, anti-human, REAfinity™

Documents and Protocols

Brochures/posters

REAfinity™ Antibodies for mass cytometry - Recombinantly engineered to increase reproducibility

Brochures/posters

REAfinity™ Recombinant Antibodies - Flow cytometry is in their genes

App notes/customer reports

Extensive characterization of NK cells using mass cytometry

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 CD56 Antibody, anti-human, REAfinity™

Publications

Camp, B. V. et al. (1990) Plasma cells in multiple myeloma express a natural killer cell- associated antigen: CD56 (NKH-1; Leu-19). Blood 76: 377-382
Vivier, E. et al. (2018) Anti-NKG2A mAb Is a Checkpoint Inhibitor that Promotes Anti-tumor Immunity by Unleashing Both T and NK Cells. Cell 175(7): 1731-1743
Boin, F. et al. (2017) Flow cytometric discrimination of seven lineage markers by using two fluorochromes. PLoS One 12(11)
Petty, R. D. et al. (2016) MicroRNAs hsa-miR-99b, hsa-miR-330, hsa-miR-126 and hsa-miR-30c: potential diagnostic biomarkers in natural killer (NK) cells of patients with chronic fatigue syndrome (CFS)/ myalgic encephalomyelitis (ME). PLoS One 11: e0150904
Dong, P. et al. (2017) Simultaneous detection of decidual Th1/Th2 and NK1/NK2 immunophenotyping in unknown recurrent miscarriage using 8-color flow cytometry with FSC/Vt extended strategy. Biosci. Rep. 37(3)
Canté-Barrett, K. et al. (2017)
Loss of CD44
^dim
expression from early progenitor cells marks T-cell lineage commitment in the human thymus.
Front Immunol 8: 32
Croci, S. et al. (2018) Higher frequencies of lymphocytes expressing the natural killer group 2D receptor in patients with Behçet disease. Front Immunol 9: 2157
Delso-Vallejo, M. et al. (2017)
Influence of irradiated peripheral blood mononuclear cells on both
ex vivo
proliferation of human natural killer cells and change in cellular property.
Front Immunol 8
Grant, M. D. et al. (2018) Natural Killer Cells Adapt to Cytomegalovirus Along a Functionally Static Phenotypic Spectrum in Human Immunodeficiency Virus Infection. Front Immunol 9: 2494
Popkin, D. L. et al. (2018)
In vitro
evidence that combination therapy with CD16-bearing NK-92 cells and FDA-approved Alefacept can selectively target the latent HIV reservoir in CD4
⁺
CD2
^hi
memory T cells.
Front Immunol 9: 2552
Soldierer, M. et al. (2022) Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies, Front Immunol 13: doi: 10.3389/fimmu.2022.847008
Veluchamy, J. et al. (2017)
In vivo
efficacy of umbilical cord blood stem cell-derived NK cells in the treatment of metastatic colorectal cancer.
Front Immunol 8: 87

Applications

Extended validation