CD54 (ICAM-1) Antibody, anti-human,
REAlease
^®

Name: CD54 (ICAM-1) Antibody, anti-human, REAlease®
Availability: InStock

CD54 (ICAM-1) Antibody, anti-human,
REAlease
^®
Extended ValidationRecombinant

Clone:

REAL146

Type of antibody:

Releasable antibodies, Primary antibodies, Recombinant antibodies

Applications:

FC, MICS, IHC, IF, FA

Alternative names:

ICAM-1, BB2, P3.58

Flow cytometry applications forCD54 (ICAM-1) Antibody, anti-human,
REAlease
^®

PE

Conjugate:

PE

Recommended dilution:

1:50

Tested:

QC tested

Products used:

130-117-841, 130-117-729, 130-124-857

Protocols:

Cell surface staining protocol using REAlease™ antibodies

Description:

Human peripheral blood mononuclear cells (PBMCs) were stained with REAlease CD54 (ICAM-1)-PE (B) or with the corresponding REAL Control (A). REAlease CD54 (ICAM-1)-PE was released using the REAlease Release Reagent (C). To show epitope accessibility after REAlease Complex release, the cells were washed with the REAlease Stop Reagent and restained with REAlease CD54 (ICAM-1)-PE (D). Cells were labeled with CD19 antibodies in addition and analyzed by flow cytometry using the MACSQuant^® Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide (PI) fluorescence.

MICS (MACSima Imaging Cyclic Staining) applications forCD54 (ICAM-1) Antibody, anti-human,
REAlease
^®

APC
PE

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

PFA

Tested:

QC tested

Species tested:

human

Products used:

130-112-099

Tissue used:

tonsil

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

Description:

Immunofluorescence analysis of a human tonsil cryosection fixed with 4% PFA. The section was stained using CD54 (ICAM-1) Antibody, anti-human, APC (green) for 10 min at room temperature (A). CD54 (ICAM-1) Antibody, anti-human, APC staining was released using the REAlease Release Reagent (B). Nuclei were visualized with Hoechst™ (blue). Scale bar: 30 µm.

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

Acetone

Tested:

during development

Species tested:

human

Products used:

130-112-099

Tissue used:

tonsil

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

Description:

Immunofluorescence analysis of a human tonsil cryosection fixed with ice-cold acetone. The section was stained using CD54 (ICAM-1) Antibody, anti-human, APC (green) for 10 min at room temperature (A). CD54 (ICAM-1) Antibody, anti-human, APC staining was released using the REAlease Release Reagent (B). Nuclei were visualized with Hoechst™ (blue). Scale bar: 30 µm.

Extended validation for CD54 (ICAM-1) Antibody, anti-human,
REAlease
^®

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 REAL146
HCD54	++
LB-2	-
HA58	++
RR1/1	++
REA266	++
Cells were incubated with an excess of purified unconjugated CD54 (ICAM-1) (REAL146) 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 CD54 (ICAM-1) knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD54 (ICAM-1)-PE (REAL146, 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 (REAL146, red) and counterstained with DRAQ5 (blue) as DNA stain.

Overlay histogram showing flow cytometric analysis of human CD54 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD54-PE, clone (REAL146). 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 (REAL146). 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 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.

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.

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.

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

Comparison of staining pattern on non-fixed and fixed cells using CD54 (ICAM-1) (REAL146). 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) (REAL146). 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,
REAlease
^®

Overview

Clone REAL146 is an antibody fragment derived from the full CD54 (ICAM-1) antibody molecule. It displays no binding to Fc receptors. The recombinantly engineered antibody fragments are multimerized to form the REAlease Complex to bind markers with high avidity.

Clone REAL146 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 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.

The REAlease Kits consist of the respective fluorochrome-conjugated REAlease Complexes and the REAlease Support Kit for removal of the REAlease Complexes and optional relabeling with different fluorochrome-conjugated REAlease Complexes.

Alternative names

ICAM-1, BB2, P3.58

Detailed product information

Technical specifications

Clone	REAL146
Clonality	monoclonal
Isotype control	Control Antibody
Host	cell line
Type of antibody	Releasable antibodies, Primary antibodies, Recombinant antibodies
Species	human
Antigen	CD54 (ICAM-1)
Alternative names of antigen	ICAM-1, BB2, P3.58
Distribution of antigen	monocytes, macrophages, lymphocytes, endothelial cells
RRID	AB_2801707, AB_2857378, AB_2811699, AB_2801709

Resources for CD54 (ICAM-1) Antibody, anti-human,
REAlease
^®

Certificates

Please follow this

link

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

References for CD54 (ICAM-1) Antibody, anti-human,
REAlease
^®

Publications

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
Greve, J. M. et al. (1989) The major human rhinovirus receptor is ICAM-1. Cell 56(5): 839-847
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

Applications

Extended validation