Description
The MC480 monoclonal antibody reacts with Stage-Specific Embryonic Antigen-1 (SSEA-1), which is a terminal carbohydrate epitope (3-fucosyl-N-acetyllactosamine or 3-FAL) on glycoproteins and lactose-series glycolipids. SSEA-1 is related to Lewis blood group antigens and is found in a variety of embryonic as well as adult tissues and cancers. As its name implies, the expression of SSEA-1 is stage-specific and can be used to characterize embryonic cells and monitor their differentiation. However, its expression pattern differs between human and mice. In the human, SSEA-1 is not found on embryonic stem (ES) cells, embryonic inner cell mass (ICM), or teratocarcinoma (embryonal carcinoma or EC) cells. As human EC and ES cells undergo differentiation, SSEA-1 expression is upregulated. In the adult, the same epitope is expressed as CD15 on granulocytes and monocytes, but not lymphocytes or dendritic cells. In the mouse, SSEA-1 is found on EC, ES, primordial germ cells, 8-cell to blastocyst embryos, ICM, and subpopulations of cells in the adult central nervous system, including stem cells. In contrast to human SSEA-1 expression, it is reduced when mouse EC and ES cells undergo differentiation.
Recommended Assay Procedure: 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 1:10 in 1× PBS, and stain the cells by adding 50 µl of the diluted antibody conjugate to each well and incubating for 1 hour at RT. 5. Remove the diluted antibody, and wash the wells twice with 100 μl of 1× PBS. 6. 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. 7. View and analyze the cells on an appropriate imaging instrument. Recommended filters for the BD Pathway™ cell analyzers are: Instrument Excitation Emission Dichroic BD Pathway 855 488/10 515 LP Fura/FITC BD Pathway 435 482/35 536/40 FF506
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 488 under optimum conditions, and unreacted Alexa Fluor® 488 was removed.
1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
2. This reagent has been pre-diluted for use at the recommended Volume per Test when following the Recommended Assay Procedure. A Test is typically ~10,000 cells cultured in a well of a 96-well imaging plate.
3. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
4. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
5. 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.
6. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
