Clone:
REA351
Type of antibody:
Primary antibodies, Recombinant antibodies
Isotype:
recombinant human IgG1
Applications:
FC, MICS, IF, IHC, MC
Alternative names:
LAG-3, Protein FDC

Extended validation for CD223 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 REA351
T47-530+
3DS223H++
11C3C65-
7H2C65+
17B4++
Cells were incubated with an excess of purified unconjugated CD223 (REA351) 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.

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 CD223. Human peripheral blood mononuclear cells (PBMCs) stimulated with CD3/CD28 antibodies (T Cell TransAct™) for 3 days were stained with CD223 antibodies and with a suitable counterstaining. As a control, CD223 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.
View details
Flow cytometric comparison of different clones for CD223. Human peripheral blood mononuclear cells (PBMCs) stimulated with CD3/CD28 antibodies (T Cell TransAct™) for 3 days were stained with CD223 antibodies and with a suitable counterstaining. As a control, CD223 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.
View details
Flow cytometric comparison of different clones for CD223. Human peripheral blood mononuclear cells (PBMCs) stimulated with CD3/CD28 antibodies (T Cell TransAct™) for 3 days were stained with CD223 antibodies and with a suitable counterstaining. As a control, CD223 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.
View details
Flow cytometric comparison of different clones for CD223. Human peripheral blood mononuclear cells (PBMCs) stimulated with CD3/CD28 antibodies (T Cell TransAct™) for 3 days were stained with CD223 antibodies and with a suitable counterstaining. As a control, CD223 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.
View details
Flow cytometric comparison of different clones for CD223. Human peripheral blood mononuclear cells (PBMCs) stimulated with CD3/CD28 antibodies (T Cell TransAct™) for 3 days were stained with CD223 antibodies and with a suitable counterstaining. As a control, CD223 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.
View details
Flow cytometric comparison of different clones for CD223. Human peripheral blood mononuclear cells (PBMCs) stimulated with CD3/CD28 antibodies (T Cell TransAct™) for 3 days were stained with CD223 antibodies and with a suitable counterstaining. As a control, CD223 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 CD223. Human peripheral blood mononuclear cells (PBMCs) stimulated with CD3/CD28 antibodies (T Cell TransAct™) for 3 days were stained with CD223 antibodies and with a suitable counterstaining. As a control, CD223 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
View details
Comparison of staining pattern on non-fixed and fixed cells using CD223 (REA351). The performance of the antibody after fixation was tested by comparing the staining pattern on fresh (no fixation) versus fixed (post fixation) cells.
View details
Comparison of staining pattern on non-fixed and fixed cells using CD223 (REA351). 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 CD223 (REA351). 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 CD223 Antibody, anti-human, REAfinity™

Overview

Clone REA351 recognizes the human CD223 antigen, a single-pass type I membrane protein also known as lymphocyte activation gene 3 protein (LAG-3). CD223 is a CD4 homolog with four extracellular Ig superfamily domains. Similar to CD4, CD223 oligomerizes at the surfaces of T cells and binds to MHC II on antigen presenting cells but with significantly higher affinity than CD4. CD223 is expressed on activated CD4
+
and CD8
+
T lymphocytes where it associates with the CD3–TCR complex at the cell surface and negatively regulates signal transduction. As a consequence, it negatively regulates T cell proliferation, function, and homeostasis. There is also increasing evidence of its involvement in regulatory function of tumor-infiltrated T cells in cancer, such as in Hodgkin’s lymphomas and in prostate cancer. CD223 is also expressed on activated NK cells, B cells, and plasmacytoid dendritic cells.
Additional information: Clone REA351 displays negligible binding to Fc receptors.

Alternative names

LAG-3, Protein FDC

Detailed product information

Technical specifications

CloneREA351
Clonalitymonoclonal
Isotyperecombinant human IgG1
Isotype controlREA Control Antibody (S), human IgG1
Hostcell line
Type of antibodyPrimary antibodies, Recombinant antibodies
Specieshuman
AntigenCD223
Alternative names of antigenLAG-3, Protein FDC
Molecular mass of antigen [kDa]54
Distribution of antigenT cells, lymphocytes
Entrez Gene ID3902
RRIDAB_2733935, AB_2751648, AB_2751602, AB_2784077, AB_2784076, AB_2801747, AB_2801740, AB_2784079, AB_2784078, AB_2819662, AB_2733934

Resources for CD223 Antibody, anti-human, REAfinity™

Certificates

Please follow this
link
to search for Certificates of Analysis (CoA) by lot number.

References for CD223 Antibody, anti-human, REAfinity™

Publications

  1. Huang, C. T. et al. (2004) Role of LAG-3 in regulatory T cells. Immunity 21(4): 503-513
  2. Triebel, F. et al. (1990) LAG-3, a novel lymphocyte activation gene closely related to CD4. J. Exp. Med. 171(5): 1393-1405
  3. Gagliani, N. et al. (2013) Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells. Nat Med 19(6): 739-746

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