Confocal immunofluorescent analysis of NTERA2 cells using SSEA4 (MC813) Mouse mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).
Flow cytometric analysis of Jurkat cells (blue) and NTERA-2 cells (green) using SSEA4 (MC813) Mouse mAb (solid lines) or a concentration-matched Mouse (G3A1) mAb IgG1 Isotype Control #5415 (dashed lines). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
Recommended Fluorochrome-conjugated Anti-Mouse secondary antibodies:
NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted December 2010
Protocol Id: 144
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If live cell staining is desired, proceed to Section C.
NOTE: Count cells using a hemocytometer or alternative method.
posted January 2009
revised June 2020
Protocol Id: 36
SSEA4 (MC813) Mouse mAb detects endogenous levels of SSEA4 antigen.
Monoclonal antibody is produced by injecting animals with human embryonal carcinoma 2102Ep cl.2A6 cells.
SSEA4 (stage-specific embryonic antigen 4) is a glycolipid carbohydrate epitope expressed on the surface of human teratocarcinoma stem cells, human embryonic germ cells, and human embryonic stem cells (1). Expression of human SSEA4 decreases following differentiation of human embryonal carcinoma cells. Expression of the SSEA4 antigen is absent in murine pluripotent cells, but increases following differentiation (1,2).
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