Immunofluorescent staining of human embryonic stem (ES) cell line. H9 cells (WiCell, Madison, WI) passage 49 were grown in mTeSR™1 Medium (StemCell Technologies) on Matrigel® hESC-qualified matrix (Corning) in a Falcon® 96 Well Imaging Plate (Corning). They were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized using BD Perm/Wash™ Perm/Wash Buffer (Cat. No. 554723), and stained with 20 µl of Alexa Fluor® 647 Mouse Anti-Human Lin-28 monoclonal antibody (pseudo-colored red) per 50 µl of stain solution per well. The cell nuclei were counterstained with DAPI (pseudo-colored blue). The images were captured on a BD Pathway™ 435 Cell Analyzer and merged using BD Attovision™ software. BD Phosflow™ Perm Buffer III (Cat. No. 558050) is also suitable for permeabilization.
Immunofluorescent staining of human embryonic stem (ES) cell line. H9 cells (WiCell, Madison, WI) passage 49 were grown in mTeSR™1 Medium (StemCell Technologies) on Matrigel® hESC-qualified matrix (Corning) in a Falcon® 96 Well Imaging Plate (Corning). They were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized using BD Perm/Wash™ Perm/Wash Buffer (Cat. No. 554723), and stained with 20 µl of Alexa Fluor® 647 Mouse Anti-Human Lin-28 monoclonal antibody (pseudo-colored red) per 50 µl of stain solution per well. The cell nuclei were counterstained with DAPI (pseudo-colored blue). The images were captured on a BD Pathway™ 435 Cell Analyzer and merged using BD Attovision™ software. BD Phosflow™ Perm Buffer III (Cat. No. 558050) is also suitable for permeabilization.
Preparation And Storage
Product Notices
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- 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.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- mTESR™1 is a trademark of StemCell Technologies.
- Accutase is a registered trademark of Innovative Cell Technologies, Inc.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Lin-28 is an evolutionarily conserved RNA-binding protein that is expressed in embryonic tissue, embryonic stem cells, and some adult tissues (e.g. cardiac and skeletal muscle). Within these cells, it is localized to ribosomes, P-bodies, and stress granules. During embryonic development, Lin-28 acts as a "translational enhancer" by binding to certain mRNAs and increasing their translation efficiency. Additionally, Lin-28 is overexpressed in some human cancer cell lines and is associated with the malignant transformation of cells as well as advanced human malignancies. This role is thought to be tied to the binding of Lin-28 to pre-microRNA of let-7, which in its mature form promotes terminal differentiation of stem cells and suppresses tumors. The binding of Lin-28 to the immature version of let-7 blocks its cleavage into mature microRNA, thereby preventing it from carrying out its function in tumor suppression. Lin-28, in combination with additional factors, has been used to reprogram human somatic cells into induced pluripotent stem cells.
Development References (7)
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Balzer E, Moss EG. Localization of the developmental timing regulator Lin28 to mRNP complexes, P-bodies and stress granules. RNA Biol. 2007; 4(1):16-25. (Biology). View Reference
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Darr H, Benvenisty N. Genetic analysis of the role of the reprogramming gene LIN-28 in human embryonic stem cells. Stem Cells. 2008. (Biology). View Reference
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Newman MA, Thomson JM, Hammond SM. Lin-28 interaction with the Let-7 precursor loop mediates regulated microRNA processing. RNA. 2008; 14(8):1539-1549. (Biology). View Reference
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Polesskaya A, Cuvellier S, Naguibneva I, Duquet A, Moss EG, Harel-Bellan A. Lin-28 binds IGF-2 mRNA and participates in skeletal myogenesis by increasing translation efficiency. Genes Dev. 2007; 21(9):1125-1138. (Biology). View Reference
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Viswanathan SR, Daley GQ, Gregory RI. Selective blockade of microRNA processing by Lin28. Science. 2008; 320:97-100. (Biology: Array). View Reference
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Viswanathan SR, Powers JT, Einhorn W, et al. Lin28 promotes transformation and is associated with advanced human malignancies. Nat Genet. 2009; 41(7):843-848. (Biology). View Reference
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Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007; 318(5858):1917-1920. (Biology). View Reference
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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
