Clone:
REA997
Type of antibody:
Primary antibodies, Recombinant antibodies
Isotype:
recombinant human IgG1
Applications:
FC, MICS, IF, IHC, MC
Alternative names:
FCGR2A, FCGR2B, FCGR2C, FcγRII

Extended validation for CD32 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 REA997
FUN-2+
FLIP8.26++
3D3+
6C4++
2E1++
Cells were incubated with an excess of purified unconjugated CD32 (REA997) 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 CD32 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD32-PE (REA997, red) and counterstained with DRAQ5 (blue) as DNA stain.
View details
Fluorescence microscopy image of CD32 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD32-PE (REA997, red) and counterstained with DRAQ5 (blue) as DNA stain.
Fluorescence microscopy image of CD32 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD32-PE (REA997, red) and counterstained with DRAQ5 (blue) as DNA stain.
View details
Overlay histogram showing flow cytometric analysis of human CD32 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD32-PE, clone (REA997). 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 CD32 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD32-PE, clone (REA997). 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 CD32. Human peripheral blood mononuclear cells (PBMCs) were stained with CD32 antibodies and with a suitable counterstaining. As a control,CD32 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 CD32. Human peripheral blood mononuclear cells (PBMCs) were stained with CD32 antibodies and with a suitable counterstaining. As a control,CD32 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 CD32. Human peripheral blood mononuclear cells (PBMCs) were stained with CD32 antibodies and with a suitable counterstaining. As a control,CD32 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 CD32. Human peripheral blood mononuclear cells (PBMCs) were stained with CD32 antibodies and with a suitable counterstaining. As a control,CD32 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 CD32. Human peripheral blood mononuclear cells (PBMCs) were stained with CD32 antibodies and with a suitable counterstaining. As a control,CD32 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 CD32. Human peripheral blood mononuclear cells (PBMCs) were stained with CD32 antibodies and with a suitable counterstaining. As a control,CD32 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 CD32 (REA997). 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 CD32 (REA997). 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 CD32 (REA997). 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 CD32 Antibody, anti-human, REAfinity™

Overview

Clone REA997 recognizes the human CD32 antigen, also known as FcγRII. CD32 is a 40 kDa low affinity receptor for the Fc fragment of aggregated IgG. Due to alternative splicing three different isoforms of CD32 are expressed on hematopoietic cells: FcRIIA, FcRIIB, and FcRIIC. The cytoplasmic domains of FcRIIA and FcRIIC contain an activatory ITAM motif whereas FcRIIB is an inhibitory receptor containing an ITIM motif. Different isoforms of CD32 are expressed on B cells, monocytes, dendritic cells, granulocytes, and platelets. Clone REA997 recognizes all isoforms of CD32 without blocking the binding of immune complexes.
Additional information: Clone REA997 displays negligible binding to Fc receptors.

Alternative names

FCGR2A, FCGR2B, FCGR2C, FcγRII

Detailed product information

Technical specifications

CloneREA997
Clonalitymonoclonal
Isotyperecombinant human IgG1
Isotype controlREA Control Antibody (S), human IgG1
Hostcell line
Type of antibodyPrimary antibodies, Recombinant antibodies
Specieshuman, non-human primate, other
Cross-reactivity
rhesus monkey (
Macaca mulatta
)
,
cynomolgus monkey (
Macaca fascicularis
)
, baboon, horse
AntigenCD32
Alternative names of antigenFCGR2A, FCGR2B, FCGR2C, FcγRII
Molecular mass of antigen [kDa]31
Distribution of antigenB cells, dendritic cells, endothelial cells, granulocytes, leukocytes, basophils, eosinophils, neutrophils, plasma cells, placenta
Entrez Gene ID2212
RRIDAB_2727663, AB_2727618, AB_2727662, AB_2727617, AB_2819613

References for CD32 Antibody, anti-human, REAfinity™

Publications

  1. Stuart, S. G. et al. (1989) Human IgG Fc receptor (hFcRII; CD32) exists as multiple isoforms in macrophages, lymphocytes and IgG-transporting placental epithelium. EMBO J. 8(12): 3657-3666
  2. Van Den Herik-Oudijk, I. E. et al. (1994) Functional analysis of human Fc gamma RII (CD32) isoforms expressed in B lymphocytes. J. Immunol. 152(2): 574-585
  3. Båve, U. et al. (2003) FcγRIIa is expressed on natural IFN-α-producing cells (plasmacytoid dendritic cells) and is required for the IFN-α production induced by apoptotic cells combined with lupus IgG. J. Immunol. 171: 3296-3302

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