Clone:
REA266
Type of antibody:
Primary antibodies, Recombinant antibodies
Isotype:
recombinant human IgG1
Applications:
FC, MICS, IF, IHC
Alternative names:
ICAM-1, BB2, P3.58

Extended validation for CD54 (ICAM-1) 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 REA266
HCD54++
LB-2-
HA58++
RR1/1++
REAL146++
Cells were incubated with an excess of purified unconjugated CD54 (ICAM-1) (REA266) 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 CD54 (ICAM-1) knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD54 (ICAM-1)-PE (REA266, red) and counterstained with DRAQ5 (blue) as DNA stain.
View details
Fluorescence microscopy image of CD54 (ICAM-1) knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD54 (ICAM-1)-PE (REA266, red) and counterstained with DRAQ5 (blue) as DNA stain.
Fluorescence microscopy image of CD54 (ICAM-1) knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD54 (ICAM-1)-PE (REA266, red) and counterstained with DRAQ5 (blue) as DNA stain.
View details
Overlay histogram showing flow cytometric analysis of human CD54 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD54-PE, clone (REA266). 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 CD54 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD54-PE, clone (REA266). 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 CD54 (ICAM-1). Human peripheral blood mononuclear cells (PBMCs) were stained with CD54 (ICAM-1)antibodies and with a suitable counterstaining. As a control, CD54 (ICAM-1) 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 CD54 (ICAM-1). Human peripheral blood mononuclear cells (PBMCs) were stained with CD54 (ICAM-1)antibodies and with a suitable counterstaining. As a control, CD54 (ICAM-1) 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 CD54 (ICAM-1). Human peripheral blood mononuclear cells (PBMCs) were stained with CD54 (ICAM-1)antibodies and with a suitable counterstaining. As a control, CD54 (ICAM-1) 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 CD54 (ICAM-1). Human peripheral blood mononuclear cells (PBMCs) were stained with CD54 (ICAM-1)antibodies and with a suitable counterstaining. As a control, CD54 (ICAM-1) 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 CD54 (ICAM-1). Human peripheral blood mononuclear cells (PBMCs) were stained with CD54 (ICAM-1)antibodies and with a suitable counterstaining. As a control, CD54 (ICAM-1) 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 CD54 (ICAM-1). Human peripheral blood mononuclear cells (PBMCs) were stained with CD54 (ICAM-1)antibodies and with a suitable counterstaining. As a control, CD54 (ICAM-1) 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 CD54 (ICAM-1). Human peripheral blood mononuclear cells (PBMCs) were stained with CD54 (ICAM-1)antibodies and with a suitable counterstaining. As a control, CD54 (ICAM-1) 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 CD54 (ICAM-1). Human peripheral blood mononuclear cells (PBMCs) were stained with CD54 (ICAM-1)antibodies and with a suitable counterstaining. As a control, CD54 (ICAM-1) 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 CD54 (ICAM-1) (REA266). 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 CD54 (ICAM-1) (REA266). 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 CD54 (ICAM-1) (REA266). 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 CD54 (ICAM-1) Antibody, anti-human, REAfinity™

Overview

Clone REA266 recognizes human CD54, a 85–110 kDa type I transmembrane glycoprotein, which also known as intercellular adhesion molecule 1 (ICAM-1). CD54 is continuously present in low concentrations in the membranes of monocytes/macrophages, lymphocytes, activated endothelial cells, granulocytes, and dendritic cells. Its expression is regulated by inflammatory cytokines and can be induced by interleukin 1 (IL-1) and tumor necrosis factor. CD54 is a ligand for LFA-1, a receptor found on leukocytes. When activated, leukocytes bind to endothelial cells via CD54/LFA-1 and then transmigrate into tissues. CD54 has been shown to interact with CD11a, EZR, and CD18. More recently, CD54 has been characterized as a site for the cellular entry of human rhinovirus.
Additional information: Clone REA266 displays negligible binding to Fc receptors.

Alternative names

ICAM-1, BB2, P3.58

Detailed product information

Technical specifications

CloneREA266
Clonalitymonoclonal
Isotyperecombinant human IgG1
Isotype controlREA Control Antibody (S), human IgG1
Hosthuman cell line
Type of antibodyPrimary antibodies, Recombinant antibodies
Specieshuman
AntigenCD54 (ICAM-1)
Alternative names of antigenICAM-1, BB2, P3.58
Molecular mass of antigen [kDa]55
Distribution of antigenendothelial cells, dendritic cells, macrophages, monocytes, lymphocytes
Entrez Gene ID3383
RRIDAB_2752175, AB_2784252, AB_2784251, AB_2658698, AB_2658699, AB_2658702, AB_2658703, AB_2921798, AB_2921792, AB_2905073, AB_2905072, AB_2922098, AB_2922069, AB_2752194

Resources for CD54 (ICAM-1) Antibody, anti-human, REAfinity™

Certificates

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

References for CD54 (ICAM-1) Antibody, anti-human, REAfinity™

Publications

  1. Simmons, D. et al. (1988) ICAM, an adhesion ligand of LFA-1, is homologous to the neural cell adhesion molecule NCAM. Nature 331(6157): 624-627
  2. Greve, J. M. et al. (1989) The major human rhinovirus receptor is ICAM-1. Cell 56(5): 839-847
  3. Shimaoka, M. et al. (2003) Structures of the α L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation. Cell 112(1): 99-111
  4. Vernot, J. P. et al. (2017)
    Phenotypic and functional alterations of hematopoietic stem and progenitor cells in an
    in vitro
    leukemia-induced microenvironment.
    Int J Mol Sci 18(2): E199

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