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Purified Mouse anti-SSEA-4
Purified Mouse anti-SSEA-4

Immunofluorescent staining of human EC and ES cell lines.  The 2102Ep cell line (clone 2/A6, Josephson et al, 2007) was obtained from Dr. Peter W. Andrews, the University of Sheffield, Sheffield, UK, and the H9 cell line (Thomson et al, 1998) was obtained from WiCell, Madison, WI.  The cells were cultured, fixed, and stained with Purified Mouse anti-SSEA-4 monoclonal antibody (pseudo-colored green) according to the Recommended Assay Procedure.  The second-step reagent was Alexa Fluor® 647 goat anti-mouse Ig (Invitrogen) and counter-staining was with Hoechst 33342 (pseudo-colored blue).  The images were captured on a BD Pathway™ 855 Cell Analyzer and merged using BD Attovision™ software.  

LEFT PANEL: 2102Ep cells, 20X.  MIDDLE PANEL: H9 cells, 20X.  RIGHT PANEL: H9 cells, 4X.

Immunofluorescent staining of human EC and ES cell lines.  The 2102Ep cell line (clone 2/A6, Josephson et al, 2007) was obtained from Dr. Peter W. Andrews, the University of Sheffield, Sheffield, UK, and the H9 cell line (Thomson et al, 1998) was obtained from WiCell, Madison, WI.  The cells were cultured, fixed, and stained with Purified Mouse anti-SSEA-4 monoclonal antibody (pseudo-colored green) according to the Recommended Assay Procedure.  The second-step reagent was Alexa Fluor® 647 goat anti-mouse Ig (Invitrogen) and counter-staining was with Hoechst 33342 (pseudo-colored blue).  The images were captured on a BD Pathway™ 855 Cell Analyzer and merged using BD Attovision™ software.  

LEFT PANEL: 2102Ep cells, 20X.  MIDDLE PANEL: H9 cells, 20X.  RIGHT PANEL: H9 cells, 4X.

Product Details
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BD Pharmingen™
Human (QC Testing), Mouse (Reported)
Mouse BALB/c IgG3, κ
Human Teratocarcinoma Cell Line
Bioimaging (Routinely Tested), Western blot (Tested During Development), Flow cytometry (Reported)
0.5 mg/ml
AB_1645601
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.

Recommended Assay Procedures

1.        Seed the cells in appropriate culture medium at an appropriate cell density in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219), and

        culture overnight to 48 hours.

2.        Remove the culture medium from the wells, wash the wells twice with 100 μl of 1× PBS, and fix the cells by adding 100 µl of fresh 3.7% Formaldehyde in PBS or BD Cytofix™ fixation buffer (Cat. No. 554655) to each well and incubating for 10 minutes at room temperature (RT).

3.        Remove the fixative from the wells, and wash the wells twice with 100 μl of 1× PBS.

4.        Dilute the antibody in 1× PBS, and stain the cells by adding 50 µl of the diluted antibody to each well and incubating for 1 hour at RT.

5.        Remove the diluted antibody, and wash the wells three times with 100 μl of 1× PBS.

6.        Remove the PBS, dilute the second-step reagent in 1× PBS, and stain the cells by adding 50 µl of the diluted second-step reagent to each well and incubating for 1 hour at RT.

7.        Remove the diluted second-step reagent, and wash the wells twice with 100 μl of 1× PBS.

8.        Remove the PBS, and counter-stain the nuclei by adding 100 μl of a 2 μg/ml solution of Hoechst 33342 (eg, Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.

9.        View and analyze the cells on an appropriate imaging instrument.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
  3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  4. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560073 Rev. 8
Antibody Details
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MC813-70

The MC813-70 monoclonal antibody reacts with Stage-Specific Embryonic Antigen-4 (SSEA-4), a carbohydrate epitope on the major ganglioside, but not the neutral glycolipid, of human teratocarcinoma cells.  As its name implies, the expression of SSEA-4 is stage-specific and can be used to characterize embryonic cells and monitor their differentiation.  However, its expression pattern differs in the human and mouse.  In the human, SSEA-4 is found on teratocarcinoma (embryonal carcinoma or EC), embryonic inner cell mass (ICM), embryonic stem (ES) cells, and the K562 erythromyeloid leukeumia cell line.  As human stem cells undergo differentiation, SSEA-4 expression is lost.  In the mouse, SSEA-4 is found on oocytes and early cleavage-stage embryos, and primitive ectoderm, but not on EC, ICM, or ES cells.  In some cases, SSEA-4 expression appears upon differentiation of mouse EC or ES cells.

560073 Rev. 8
Format Details
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
560073 Rev.8
Citations & References
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Development References (6)

  1. Draper JS, Pigott C, Thomson JA, Andrews PW. Surface antigens of human embryonic stem cells: changes upon differentiation in culture. J Anat. 2002; 200:249-258. (Clone-specific: Flow cytometry). View Reference
  2. Henderson JK, Draper JS, Baillie HS, et al. Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens. Stem Cells. 2002; 20:329-337. (Clone-specific: Flow cytometry, Immunocytochemistry (cytospins)). View Reference
  3. Josephson R, Ording CJ, Liu Y, et al. Qualification of embryonal carcinoma 2102Ep as a reference for human embryonic stem cell research. Stem Cells. 2007; 25:437-446. (Clone-specific: Flow cytometry, Immunofluorescence). View Reference
  4. Kannagi R, Cochran NA, Ishigami F, et al. Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells. EMBO J. 1983; 2(12):2355-2361. (Immunogen: Immunofluorescence, Radioimmunoassay). View Reference
  5. Son YS, Park JH, Kang YK, et al. Heat shock 70-kDa protein 8 isoform 1 is expressed on the surface of human embryonic stem cells and downregulated upon differentiation. Stem Cells. 2005; 23:1502-1513. (Clone-specific: Flow cytometry, Immunocytochemistry (cytospins)). View Reference
  6. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998; 282:1145-1147. (Clone-specific: Immunocytochemistry (cytospins)). View Reference
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560073 Rev. 8

Please refer to Support Documents for Quality Certificates


Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product as described.

Comparisons, where applicable, are made against older BD technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.