F4/80 Antibody, anti-mouse, REAfinity™

Name: F4/80 Antibody, anti-mouse, REAfinity™
Availability: InStock

F4/80 Antibody, anti-mouse, REAfinity™Extended ValidationRecombinant

Clone:

REA126

Type of antibody:

Primary antibodies, Recombinant antibodies

Isotype:

recombinant human IgG1

Applications:

FC, MICS, IF, IHC, 3D-IF

Alternative names:

ADGRE1, DD7A5-7, EGF-TM7, EMR1, Gpf480, Ly71, TM7LN3

Flow cytometry applications forF4/80 Antibody, anti-mouse, REAfinity™

APC
Biotin
FITC
PE
PE-Vio 615
PE-Vio 770
PerCP-Vio 700

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:

130-116-525, 130-116-547

Protocols:

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

Description:

Spleen cells from BALB/c mouse were stained with Anti-F4/80 antibodies as well as with Anti-Ly-6C antibodies and analyzed by flow cytometry using the MACSQuant^® Analyzer. CD45.2⁺ cells were pre-gated for the analysis. 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.

MICS (MACSima Imaging Cyclic Staining) applications forF4/80 Antibody, anti-mouse, REAfinity™

APC
FITC
PE

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

PFA

Tested:

during development

Species tested:

mouse

Products used:

130-116-525, 130-116-547

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

Acetone

Tested:

during development

Species tested:

mouse

Products used:

130-116-525, 130-116-547

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

3D-Immunofluorescence applications forF4/80 Antibody, anti-mouse, REAfinity™

Vio R667

Conjugate:

Vio R667

Recommended dilution:

1:50

Fixation:

PFA

Tested:

during development

Species tested:

mouse

Products used:

130-131-632

Tissue clearing:

MACS^® Clearing Kit

Compatible applications:

IF, IHC

Protocols:

Immunostaining and clearing of mouse brain hemispheres for 3D imaging analysis

Extended validation for F4/80 Antibody, anti-mouse, 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 REA126
BM8	++
CI:A3-1	-
T45-2342	-
Cells were incubated with an excess of purified unconjugated F4/80 (REA126) 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.

Flow cytometric comparison of different clones for F4/80. Peritoneal macrophages from C57BL/6 mice were stained with F4/80 antibodies and with a suitable counterstaining. As a control, F4/80 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 F4/80. Peritoneal macrophages from C57BL/6 mice were stained with F4/80 antibodies and with a suitable counterstaining. As a control, F4/80 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 F4/80. Peritoneal macrophages from C57BL/6 mice were stained with F4/80 antibodies and with a suitable counterstaining. As a control, F4/80 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 F4/80. Peritoneal macrophages from C57BL/6 mice were stained with F4/80 antibodies and with a suitable counterstaining. As a control, F4/80 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 F4/80. Peritoneal macrophages from C57BL/6 mice were stained with F4/80 antibodies and with a suitable counterstaining. As a control, F4/80 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 F4/80 (REA126). 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 F4/80 (REA126). 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 F4/80 Antibody, anti-mouse, REAfinity™

Overview

Clone REA126 recognizes F4/80, a member of epidermal growth factor (EGF)-transmembrane 7 (TM7) family. F4/80 is considered as one of the most specific cell-surface markers for murine macrophages. F4/80 is a seven-span transmembrane molecule with a large extracellular, multiple EGF module–containing domain. Constitutive and high expression of F4/80 is found on most resident tissue macrophages, including the spleen, microglia in the brain, Kupffer's cells in the liver, and Langerhans cells in the skin. In addition, the expression of F4/80 can also be regulated depending on the physiological status of the cell. F4/80 molecule is required for the differentiation of antigen-specific CD8

⁺

Tregs and is involved in inducing peripheral immune tolerance. No specific ligand for F4/80 has been reported so far.

Additional information: Clone REA126 displays negligible binding to Fc receptors.

Alternative names

ADGRE1, DD7A5-7, EGF-TM7, EMR1, Gpf480, Ly71, TM7LN3

Detailed product information

Technical specifications

Clone	REA126
Clonality	monoclonal
Isotype	recombinant human IgG1
Isotype control	REA Control Antibody, human IgG1
Host	human cell line
Type of antibody	Primary antibodies, Recombinant antibodies
Species	mouse
Antigen	F4/80
Alternative names of antigen	ADGRE1, DD7A5-7, EGF-TM7, EMR1, Gpf480, Ly71, TM7LN3
Molecular mass of antigen [kDa]	99
Distribution of antigen	macrophages
Entrez Gene ID	13733
RRID	AB_2727574, AB_2733417, AB_2733418, AB_2733260, AB_2733261, AB_2751520, AB_2751483, AB_2784335, AB_2819539, AB_2727970

Resources for F4/80 Antibody, anti-mouse, REAfinity™

Certificates

Please follow this

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

References for F4/80 Antibody, anti-mouse, REAfinity™

Publications

Lin, H.-H. et al. (2005) The macrophage F4/80 receptor is required for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance. J. Exp. Med. 201: 1615-1625
Gordon, S. et al. (2011) F4/80 and the related adhesion-GPCRs. Eur. J. Immunol. 41(9): 2472-2476
Kortlever, R. M. et al. (2017) Myc Cooperates with Ras by Programming Inflammation and Immune Suppression. Cell 171(6): 1301-1315
Hume, D. A. et al. (1984) The mononuclear phagocyte system of the mouse defined by immunohistochemical localization of antigen F4/80: macrophages of bone and associated connective tissue. J. Cell. Sci. 66: 189-194
Gu, C. et al. (2018) EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment. PLoS Pathog. 14(6)
Siegmund, D. et al. (2016) Activation of TNFR2 sensitizes macrophages for TNFR1-mediated necroptosis. Cell Death Dis 7(9): e2375
Siegmund, D. et al. (2018) TNFR2 unlocks a RIPK1 kinase activity-dependent mode of proinflammatory TNFR1 signaling. Cell Death Dis 9(9)
Lee, H. et al. (2017) Kinetics of corneal antigen presenting cells in experimental dry eye disease. BMJ Open Ophthalmol 1(1)
Jia, J. et al. (2018) Cholesterol metabolism promotes B-cell positioning during immune pathogenesis of chronic obstructive pulmonary disease. EMBO Mol. Med. 10(5)

Applications

Extended validation