CD8 Antibody, anti-human,
REAlease
^®

Name: CD8 Antibody, anti-human, REAlease®
Availability: InStock

CD8 Antibody, anti-human,
REAlease
^®
Extended ValidationRecombinant

Clone:

REAL100

Type of antibody:

Releasable antibodies, Primary antibodies, Recombinant antibodies

Applications:

FC, MICS, IHC, IF

Alternative names:

Leu-2, CD8a, MAL, p32, Cd8b1, LY3, Lyt3, P37, Ly-2

Flow cytometry applications forCD8 Antibody, anti-human,
REAlease
^®

FITC
VioGreen

Conjugate:

FITC

Recommended dilution:

1:50

Tested:

QC tested

Products used:

130-112-244, 130-112-070, 130-122-718

Protocols:

Cell surface staining protocol using REAlease™ antibodies

Description:

Human peripheral blood mononuclear cells (PBMCs) were stained with REAlease CD8-FITC (B) or with the corresponding REAL Control (A). REAlease CD8-FITC 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 CD8-FITC (D). Cells were labeled with CD3 antibodies in addition and were 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 forCD8 Antibody, anti-human,
REAlease
^®

FITC

Conjugate:

FITC

Recommended dilution:

1:50

Fixation:

Acetone

Tested:

QC tested

Species tested:

human

Products used:

130-112-244, 130-112-070

Tissue used:

tonsil

Protocols:

Protocol for MICS experiments

Description:

Immunofluorescence analysis of a human tonsil cryosection fixed with ice-cold acetone. The section was stained using CD8 Antibody, anti-human, FITC (green) for 10 min at room temperature (A). CD8 Antibody, anti-human, FITC staining was released using the REAlease Release Reagent (B). Nuclei were visualized with Hoechst™ (blue). Scale bar: 30 µm.

Extended validation for CD8 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 REAL100
OKT8	++
HIT8a	++
REA734	++
Cells were incubated with an excess of purified unconjugated CD8 (REAL100) 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 CD8. Human peripheral blood mononuclear cells (PBMCs) were stained with CD8 antibodies and with a suitable counterstaining. As a control, CD8 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 CD8. Human peripheral blood mononuclear cells (PBMCs) were stained with CD8 antibodies and with a suitable counterstaining. As a control, CD8 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 CD8. Human peripheral blood mononuclear cells (PBMCs) were stained with CD8 antibodies and with a suitable counterstaining. As a control, CD8 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 CD8. Human peripheral blood mononuclear cells (PBMCs) were stained with CD8 antibodies and with a suitable counterstaining. As a control, CD8 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 CD8. Human peripheral blood mononuclear cells (PBMCs) were stained with CD8 antibodies and with a suitable counterstaining. As a control, CD8 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 CD8. Human peripheral blood mononuclear cells (PBMCs) were stained with CD8 antibodies and with a suitable counterstaining. As a control, CD8 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 CD8. Human peripheral blood mononuclear cells (PBMCs) were stained with CD8 antibodies and with a suitable counterstaining. As a control, CD8 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 CD8 (REAL100). 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 CD8 (REAL100). 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 CD8 Antibody, anti-human,
REAlease
^®

Overview

Clone REAL100 is an antibody fragment derived from the full CD8 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 REAL100 recognizes the alpha chain of the human CD8 antigen, a cell-surface glycoprotein also known as Leu-2 or CD8a. CD8 is strongly expressed on human cytotoxic T cells and thymocytes as well as on a subset of NK cells. The CD8 antigen is a disulfide-linked dimer that exists either as a CD8α homodimer or as a CD8α/β heterodimer. CD8 acts as a co-receptor for the T cell receptor and binds to the MHC class I molecules. CD8 is involved in T cell development and activation of mature T cells.

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

Leu-2, CD8a, MAL, p32, Cd8b1, LY3, Lyt3, P37, Ly-2

Detailed product information

Technical specifications

Clone	REAL100
Clonality	monoclonal
Isotype control	Control Antibody
Host	cell line
Type of antibody	Releasable antibodies, Primary antibodies, Recombinant antibodies
Species	human
Antigen	CD8
Alternative names of antigen	Leu-2, CD8a, MAL, p32, Cd8b1, LY3, Lyt3, P37, Ly-2
Distribution of antigen	T cells, NK cells
RRID	AB_2751064, AB_2751258, AB_2751252, AB_2784285, AB_2784286, AB_2751088

Resources for CD8 Antibody, anti-human,
REAlease
^®

Certificates

Please follow this

link

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

References for CD8 Antibody, anti-human,
REAlease
^®

Publications

Devine, L. et al. (1999) Orientation of the Ig domains of CD8 alpha beta relative to MHC class I. J Immunol 162(2): 846-851
Gao, G. F. et al. (2000) Molecular interactions of coreceptor CD8 and MHC class I: the molecular basis for functional coordination with the T-cell receptor. Immunol. Today 21(12): 630-636

Applications

Extended validation