Description
The TRA-1-60 monoclonal antibody reacts with the neuraminidase-resistant form of a pluripotent-stem-cell-specific epitope on a high-molecular-weight transmembrane glycoprotein. The TRA-1-60 antigen is a sialylated epitope on the same keratan sulfate core molecule, podocalyxin, as 4 other distinct antigens on tumor-derived cell lines, TRA-1-81, GCTM2, K4, and K21. The expression of TRA-1-60 antigen is stage-specific and can be used to characterize embryonic cells and monitor their differentiation. The antigen is found on teratocarcinoma (embryonal carcinoma or EC), embryonic inner cell mass (but not morula or trophoblast), and embryonic stem (ES) cells. TRA-1-60 antigen is released into the serum of patients bearing testicular tumors containing EC cells. As human EC and ES cells undergo differentiation, expression of TRA-1-60 antigen is lost. Expression of TRA-1-60 antigen has also been observed on a rhesus monkey ES cell line (Thomson et al, 1995).
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. 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.
2. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
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. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
