Confocal immunofluorescent analysis of NTERA-2 cells, untreated (left panel) or retinoic acid-treated (10 µM all-trans RA for 5 days) (right panel), using Neurofilament-L (C28E10) Rabbit mAb #2837 (green, upper), and β3-Tubulin (TU-20) Mouse mAb #4466 (green, lower). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). Note the appearance of neuronal markers and structures as cells differentiate along the neuronal lineage with retinoic acid treatment.
Confocal immunofluorescent analysis of NTERA-2 (left) and HeLa cells (right) using Oct-4A (C30A3) Rabbit mAb (green, upper), Sox2 (D6D9) XP® Rabbit mAb (green, middle) and Nanog (D73G4) XP® Rabbit mAb (green, lower).
Projected confocal z-stack of human iPS cells using TRA-1-60(S) (TRA-1-60(S)) Mouse mAb (green, upper left), TRA-1-81 (TRA-1-81) Mouse mAb (green, upper middle), SSEA4 (MC813) Mouse mAb (green, upper right), Oct-4A (C30A3) Rabbit mAb (green, lower left), Sox2 (D6D9) XP® Rabbit mAb (green, lower middle) and Nanog (D73G4) XP® Rabbit mAb (green, lower right). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Confocal immunofluorescent analysis of NTERA-2 cells, untreated (left panel) or retinoic acid-treated (10 µM all-trans RA for 5 days) (right panel), using TRA-1-60(S) (TRA-1-60(S)) Mouse mAb (green, upper), TRA-1-81 (TRA-1-81) Mouse mAb (green, middle) and SSEA4 (MC813) Mouse mAb (green, lower). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). Note the loss of pluripotency markers (green) as cells differentiate along the neuronal lineage with retinoic acid treatment.
|Oct-4A (C30A3) Rabbit mAb||100 tests||IF-IC||1:200||Rabbit IgG|
|Sox2 (D6D9) Rabbit mAb||100 tests||IF-IC||1:200||Rabbit IgG|
|Nanog (D73G4) XP® Rabbit mAb||100 tests||IF-IC||1:200||Rabbit IgG|
|SSEA4 (MC813) Mouse mAb||100 tests||IF-IC||1:200||Mouse IgG3|
|TRA-1-60(S) (TRA-1-60(S)) Mouse mAb||100 tests||IF-IC||1:200||Mouse IgM|
|TRA-1-81 (TRA-1-81) Mouse mAb||100 tests||IF-IC||1:200||Mouse IgM|
Applications Key: IF-IC=Immunofluorescence (Immunocytochemistry)
The StemLight™ Pluripotency Antibody Kit contains a panel of antibodies for the detection of proteins that are specifically expressed in human pluripotent cells. The kit can be used to track the pluripotent potential of human embryonic stem (ES) or induced pluripotent (iPS) cells. The loss of these markers indicates a loss of pluripotency or differentiation of the culture. The kit components are pre-optimized for parallel use in immunofluorescent analysis. Enough reagents are provided for 100 assays based on a working volume of 100 µl.
Each antibody in the StemLight™ Pluripotency Antibody Kit detects endogenous levels of their respective human pluripotency marker proteins.
Nanog antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human nanog protein. Oct-4A antibody is produced by immunizing animals with recombinant protein specific to the amino terminus of human Oct-4A. Sox2 antibody is produced by immunizing animals with a synthetic peptide corresponding to amino acid sequences at the amino terminus of human Sox2. SSEA4, TRA-1-81, and TRA-1-60(S) antibodies are produced by immunizing animals with human embryonal carcinoma 2102Ep cl.2A6 cells.
Pluripotency is the ability of a cell to differentiate into cell types of the three germ layers, the endoderm, ectoderm and mesoderm. It is a property shared by embryonic stem cells, embryonic carcinoma and induced pluripotent cells.
Oct-4, Sox2 and Nanog are key transcriptional regulators that are highly expressed in pluripotent cells (1). Together they form a transcriptional network that maintains cells in a pluripotent state (2,3). Over-expression of Oct-4 and Sox2 along with Klf4 and c- Myc can induce pluripotency in both mouse and human somatic cells, highlighting their roles as key regulators of the transcrip- tional network necessary for renewal and pluripotency (4-5). It has also been demonstrated that overexpression of Oct-4, Sox2, Nanog and Lin28 can induce pluripotency in human somatic cells (6). Upon differentiation of pluripotent cultures, expression of Oct-4, Nanog and Sox2 is downregulated.
SSEA4, TRA-1-81 and TRA-1-60 antibodies recognize antigens expressed on the cell surface of all pluripotent cells. SSEA4 recognizes a glycolipid carbohydrate epitope (7). TRA-1-60(S) and TRA-1-81 antibodies recognize different proteoglycan epitopes on variants of the same protein, podocalyxin (8). These epitopes are neuraminadase sensitive and resistant, respectively. Reactivity of SSEA4, TRA-1-81 and TRA-1-60 antibodies with their respective cell surface markers are lost upon differentiation of pluripotent cells, corresponding with a loss of pluripotent potential (9).
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