CD95 (FAS) Antibody, anti-human, REAfinity™

Name: CD95 (FAS) Antibody, anti-human, REAfinity™
Availability: InStock

CD95 (FAS) Antibody, anti-human, REAfinity™Extended ValidationRecombinant

Clone:

REA738

Type of antibody:

Primary antibodies, Recombinant antibodies

Isotype:

recombinant human IgG1

Applications:

FC, MICS, IF, IHC, MC

Alternative names:

FAS, ALPS1A, APO-1, Apt1, FAS1, FASTM, Tnfrsf6

Flow cytometry applications forCD95 (FAS) Antibody, anti-human, REAfinity™

APC
APC-Vio 770
Biotin
FITC
PE
PE-Vio 615
PE-Vio 770
PerCP-Vio 700
Vio Bright B515
Vio Bright R720
Vio Bright V423

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:

130-113-070, 130-113-005

Protocols:

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

Description:

Human peripheral blood mononuclear cells (PBMCs) were stained with CD95 (FAS) antibodies or with the corresponding REA Control (S) antibodies (left images) as well as with CD45RO antibodies. Flow cytometry was performed using the MACSQuant^® Analyzer. The Tandem Signal Enhancer has been used to increase binding specificity of tandem-dye–conjugated antibodies. 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 forCD95 (FAS) Antibody, anti-human, REAfinity™

APC
FITC
PE

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

PFA

Tested:

during development

Species tested:

human

Products used:

130-113-070, 130-113-005

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

Acetone

Tested:

during development

Species tested:

human

Products used:

130-113-070, 130-113-005

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

Mass cytometry applications forCD95 (FAS) Antibody, anti-human, REAfinity™

pure

Conjugate:

pure

Products used:

130-124-328

Extended validation for CD95 (FAS) 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 clones	Overlap in epitope recognition with REA738
DX2	++
EOS9.1	-
LT95	++
4F8H6	-
SM1/23	++
Cells were incubated with an excess of purified unconjugated CD95 (FAS) (REA738) 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 CD95 (FAS) knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD95 (FAS)-PE (REA738, red) and counterstained with DRAQ5 (blue) as DNA stain.

Fluorescence microscopy image of CD95 (FAS) knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD95 (FAS)-PE (REA738, red) and counterstained with DRAQ5 (blue) as DNA stain.

Overlay histogram showing flow cytometric analysis of CD95 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD95-PE, clone (REA738). 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 CD95 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD95-PE, clone (REA738). 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 CD95 (FAS). Human peripheral blood mononuclear cells (PBMCs) were stained with CD95 (FAS)antibodies and with a suitable counterstaining. As a control, CD95 (FAS)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 CD95 (FAS). Human peripheral blood mononuclear cells (PBMCs) were stained with CD95 (FAS)antibodies and with a suitable counterstaining. As a control, CD95 (FAS)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 CD95 (FAS). Human peripheral blood mononuclear cells (PBMCs) were stained with CD95 (FAS)antibodies and with a suitable counterstaining. As a control, CD95 (FAS)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 CD95 (FAS). Human peripheral blood mononuclear cells (PBMCs) were stained with CD95 (FAS)antibodies and with a suitable counterstaining. As a control, CD95 (FAS)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 CD95 (FAS). Human peripheral blood mononuclear cells (PBMCs) were stained with CD95 (FAS)antibodies and with a suitable counterstaining. As a control, CD95 (FAS)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 CD95 (FAS). Human peripheral blood mononuclear cells (PBMCs) were stained with CD95 (FAS)antibodies and with a suitable counterstaining. As a control, CD95 (FAS)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 CD95 (FAS). Human peripheral blood mononuclear cells (PBMCs) were stained with CD95 (FAS)antibodies and with a suitable counterstaining. As a control, CD95 (FAS)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 CD95 (FAS) (REA738). 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 CD95 (FAS) (REA738). 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 CD95 (FAS) (REA738). 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 CD95 (FAS) (REA738). 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 CD95 (FAS) Antibody, anti-human, REAfinity™

Overview

Clone REA738 recognizes the human CD95 antigen, also known as Fas and Apo-1, which is a member of the tumor necrosis factor receptor superfamily (TNFR) and is found on the surface of many normal and neoplastically transformed cells. Its ligand, CD95L (FasL/Apo-1L), is able to induce apoptosis in CD95-expressing cells upon binding. CD95 and CD95L are up-regulated on lymphocytes upon activation and are known to play a key role in the regulation of an inflammatory response: Juxtocrine “fratricide” of neighbouring lymphocytes via mutual CD95 and CD95L expression helps to terminate immune responses, while apoptosis of pro-inflammatory cells via CD95 helps maintain immune privilege in sites such as the eye, where CD95L is found to be expressed in the retina and cornea. Cross-linking of CD95 receptors by DX2 monoclonal antibody has been described to induce apoptosis in certain target cells.

Additional information: Clone REA738 displays negligible binding to Fc receptors.

Alternative names

FAS, ALPS1A, APO-1, Apt1, FAS1, FASTM, Tnfrsf6

Detailed product information

Technical specifications

Clone	REA738
Clonality	monoclonal
Isotype	recombinant human IgG1
Isotype control	REA Control Antibody (S), human IgG1
Host	human cell line
Type of antibody	Primary antibodies, Recombinant antibodies
Species	human, non-human primate
Cross-reactivity	cynomolgus monkey ( Macaca fascicularis ) , rhesus monkey ( Macaca mulatta ) , african green monkey ( Chlorocebus aethiops ) , baboon, capuchin monkey, chimpanzee ( Pan troglodytes ) , common marmoset ( Callithrix jacchus ) , cotton-top tamarin ( Saguinus oedipus ) , pigtail monkey ( Macaca nemestrina ) , sooty mangabey ( Cercocebus atys )
Antigen	CD95 (FAS)
Alternative names of antigen	FAS, ALPS1A, APO-1, Apt1, FAS1, FASTM, Tnfrsf6
Molecular mass of antigen [kDa]	35
Distribution of antigen	lymphocytes
Entrez Gene ID	355
RRID	AB_2725919, AB_2784323, AB_2784322, AB_2725929, AB_2725914, AB_2811654, AB_2921880, AB_2921868, AB_2921933, AB_2921908, AB_2725915, AB_2725931, AB_2725916, AB_2725932, AB_2725917, AB_2725934, AB_2725920, AB_2725933, AB_2725918, AB_2725930

Resources for CD95 (FAS) Antibody, anti-human, REAfinity™

Certificates

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References for CD95 (FAS) Antibody, anti-human, REAfinity™

Publications

Griffith, T. et al. (1995) Fas ligand-induced apoptosis as a mechanism of immune privilege. Science 270: 1189-1192
Lynch, D. et al. (1995) Fas and FasL in the homeostatic regulation of immune responses. Immunol. Today 16: 569-574
Pitcher, C. J. et al. (2002)
Development and homeostasis of T cell memory in
rhesus macaque
.
J Immunol 168: 29-43

Applications

Extended validation