Description
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.
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.
Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
2. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
3. 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.
4. 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.
5. Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.
6. For U.S. patents that may apply, see bd.com/patents.
