Pluripotency Marker Expression in iPSCs

iPSCs at passages 15–20 were grown to confluence in 48-well tissue culture plates on mouse feeders. Cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Indirect immunofluorescence staining was then performed with antibodies against OCT3/4 (Abcam), SOX2 (R&D systems), SSEA3 (Millipore) and NANOG (Abcam), followed by Alexa fluorochrome-conjugated secondary antibodies (Life Technologies). Results were observed using Nikon Eclipse Ti-U inverted microscope with DS-Qi1 monochrome digital camera. The procedure used to stain for HNF4a and Albumin expression has been described in detail previously [28 (link)].

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Yang W., Liu Y., Slovik K.J., Wu J.C., Duncan S.A., Rader D.J, & Morrisey E.E. (2015). Generation of iPSCs as a Pooled Culture Using Magnetic Activated Cell Sorting of Newly Reprogrammed Cells. PLoS ONE, 10(8), e0134995.

Publication 2015

OCT3/4 SSEA3 NANOG Eclipse Ti-U inverted microscope DS-Qi1 monochrome digital camera

Albumin Antibodies Camera Cells Fluorochrome Indirect immunofluorescence Ipscs Microscope Mouse Oct3 Paraformaldehyde Sox2 Ssea3 Stain Tissue Triton x 100

Corresponding Organization : Cardiovascular Institute of the South

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Variable analysis

independent variables

IPSCs at passages 15–20

dependent variables

Expression of OCT3/4, SOX2, SSEA3, and NANOG

control variables

Growth of iPSCs to confluence in 48-well tissue culture plates on mouse feeders
Fixation of cells in 4% paraformaldehyde
Permeabilization of cells with 0.2% Triton X-100
Indirect immunofluorescence staining with antibodies against OCT3/4, SOX2, SSEA3, and NANOG

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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