Reprogramming assays were performed essentially as described previously9 . Briefly, dH1f fibroblasts were seeded at 2.5 × 105 cells/well in a 12-well plate and transduced overnight with a pool of lentiviral (OCT4, SOX2, NANOG) and retroviral (LIN28A variants) particles. Six days later, cells were trypsinized and 1–2 × 105 cells/well were re-plated onto MEF-coated 12-well plates. Medium was switched to hESC medium and changed daily until day 21 when reprogramming efficiency was measured. To do so, cells were fixed with 4% paraformaldehyde and stained with biotin-anti-TRA-1-60 (eBioscience #13-8863-82; 1:250) and streptavidin-HRP (Biolegend #405210; 1:500) primary and secondary antibodies, respectively. Staining was developed with the DAB Peroxidase kit (Vector Labs), and the number of iPSC colonies was quantified using ImageJ. Experiments were carried out and analyzed in a blinded manner.
Biotin anti tra 1 60
Manufactured by Thermo Fisher Scientific
Biotin-anti-TRA-1-60 is a monoclonal antibody that binds to the TRA-1-60 antigen. TRA-1-60 is a marker of undifferentiated human embryonic stem cells and induced pluripotent stem cells. Biotin-conjugation allows for detection and enrichment of TRA-1-60 positive cells.
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2 protocols using biotin anti tra 1 60
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Reprogramming Fibroblasts to iPSCs
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Reprogramming Fibroblasts to iPSCs
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Reprogramming assays were performed essentially as described previously9 . Briefly, dH1f fibroblasts were seeded at 2.5 × 105 cells/well in a 12-well plate and transduced overnight with a pool of lentiviral (OCT4, SOX2, NANOG) and retroviral (LIN28A variants) particles. Six days later, cells were trypsinized and 1–2 × 105 cells/well were re-plated onto MEF-coated 12-well plates. Medium was switched to hESC medium and changed daily until day 21 when reprogramming efficiency was measured. To do so, cells were fixed with 4% paraformaldehyde and stained with biotin-anti-TRA-1-60 (eBioscience #13-8863-82; 1:250) and streptavidin-HRP (Biolegend #405210; 1:500) primary and secondary antibodies, respectively. Staining was developed with the DAB Peroxidase kit (Vector Labs), and the number of iPSC colonies was quantified using ImageJ. Experiments were carried out and analyzed in a blinded manner.
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