SSEA-4 Antibody, anti-human, REAfinity™

Name: SSEA-4 Antibody, anti-human, REAfinity™
Availability: InStock

SSEA-4 Antibody, anti-human, REAfinity™Extended ValidationRecombinant

Clone:

REA101

Type of antibody:

Primary antibodies, Recombinant antibodies

Isotype:

recombinant human IgG1

Applications:

FC

Flow cytometry applications forSSEA-4 Antibody, anti-human, REAfinity™

APC
APC-Vio 770
Biotin
FITC
PE
PE-Vio 770
Vio Bright R720
VioBlue
VioGreen

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:

130-124-018, 130-123-815

Protocols:

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

Description:

Human induced pluripotent stem (iPS) cells were either left unstained (left peak) or stained with Anti-SSEA-4 antibodies and analyzed by flow cytometry 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.

Extended validation for SSEA-4 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 REA101
MC813	++
Cells were incubated with an excess of purified unconjugated SSEA-4 (REA101) 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 SSEA-4. NTERA-2 cells were stained with SSEA-4 antibodies and plotted against the side scatter. As a control, SSEA-4 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 SSEA-4. NTERA-2 cells were stained with SSEA-4 antibodies and plotted against the side scatter. As a control, SSEA-4 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 SSEA-4. NTERA-2 cells were stained with SSEA-4 antibodies and plotted against the side scatter. As a control, SSEA-4 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 SSEA-4 (REA101). 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 SSEA-4 (REA101). 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 SSEA-4 Antibody, anti-human, REAfinity™

Overview

Clone REA101 recognizes the glycolipid carbohydrate epitope stage specific embryonic antigen 4 (SSEA-4). SSEA-4 is found on undifferentiated human embryonic stem (ES) cells, induced pluripotent (iPS) cells, embryonal carcinoma (EC) cells, as well as embryonic germ (EG) cells and a variety of somatic stem cells, such as dental pulp stem cells, umbilical cord blood–derived very small embryonic like stem cells (VSELs), and mesenchymal stromal cells.

Additional information: Clone REA101 displays negligible binding to Fc receptors.

Detailed product information

Technical specifications

Clone	REA101
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, mouse, other
Cross-reactivity	mouse, rabbit, canine, chicken
Antigen	SSEA-4
Distribution of antigen	stem cells, ES and iPS cells
RRID	AB_2811405, AB_2811416, AB_2811404, AB_2905393, AB_2905392, AB_2877030, AB_2877031, AB_2889754, AB_2889753, AB_2876944, AB_2876945, AB_2653521, AB_2653528, AB_2921856, AB_2921840, AB_2811417

Resources for SSEA-4 Antibody, anti-human, REAfinity™

Documents and Protocols

App notes/customer reports

Multicolor flow cytometry analysis of human pluripotent stem cell cultures

Certificates

Please follow this

link

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

References for SSEA-4 Antibody, anti-human, REAfinity™

Publications

Gang, E. J. et al. (2007) SSEA-4 identifies mesenchymal stem cells from bone marrow. Blood 109(4): 1743-1751
Kannagi, R. et al. (1983) Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside. EMBO J. 2(12): 2355-2361
Truong, T. T. et al. (2011) SSEA4 is a potential negative marker for the enrichment of human corneal epithelial stem/progenitor cells. Invest. Ophthalmol. Vis. Sci. 52(9): 6315-6320
Aloia, A. et al. (2015) The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features. Breast Cancer Res. 17: 146

Applications

Extended validation