Cultured iPSCs were transferred onto Matrigel-coated 6-well plates containing DMEM/GlutaMAX (Life Technologies) supplemented with 10% fetal bovine serum and a Rho kinase inhibitor, Y-27632 (10 mM prepared in 100 μl of Dulbecco’s phosphate-buffered saline) (Sigma-Aldrich). After the cells adhered, the medium was changed to chemically defined Essential 8 medium (Life Technologies). The medium was changed daily, and cells were passaged every 5–6 days using Accutase Cell Detachment Solution (STEMCELL Technologies, Vancouver, Canada)35 (link).
The iPSC clones were fixed with 4% paraformaldehyde and immunostaining was performed using the following primary antibodies: Sox2, Tra-1–60, SSEA-4 and Oct4 and Alexa Fluor 594-conjugates or 488-conjugated secondary antibody (Life technologies). Alkaline phosphatase live cell staining was also performed (Life Technologies). These factors all stained positively in iPSC clones which were detected by indirect immunofluorescence microscopy (Carl Zeiss, Göttingen, Germany; Supplementary FigureS2D ). Cell viability and proliferation after UBC KD were assessed by crystal violet staining assay. Cells were stained with crystal violet (Sigma-Aldrich) at the indicated time points.
The iPSC clones were fixed with 4% paraformaldehyde and immunostaining was performed using the following primary antibodies: Sox2, Tra-1–60, SSEA-4 and Oct4 and Alexa Fluor 594-conjugates or 488-conjugated secondary antibody (Life technologies). Alkaline phosphatase live cell staining was also performed (Life Technologies). These factors all stained positively in iPSC clones which were detected by indirect immunofluorescence microscopy (Carl Zeiss, Göttingen, Germany; Supplementary Figure
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