af2018 r d systems eomes if  (R&D Systems)

Journal: Cell transplantation

Article Title: Endoderm and Hepatic Progenitor Cells Engraft in the Quiescent Liver Concurrent with Intrinsically Activated Epithelial-to-Mesenchymal Transition.

doi: 10.1177/0963689721993780

Figure Lengend Snippet: Figure 1. Human pluripotent stem cells efficiently form endoderm and hepatic progenitor cells in vitro. (A) RT-qPCR analysis of RNA extracted from UD, EP, HP, and ectoderm (ECT) cells displayed as delta-delta Ct values relative to HMBS “housekeeping gene product” (n ¼ 3 biological replicates shown as individual points, bars show mean, and error bars show standard deviation). (B) Representative images of indirect immunofluorescence of UD H9 ESCs showing SOX2 (magenta), SSEA3 (yellow), DAPI (blue), and each channel overlaid; of EP cells showing SOX17 (magenta), DAPI (blue), and the two channels overlaid; and HP cells with AFP (magenta), DAPI (blue), and each channel overlaid. Scale bar denotes 100 mm. (C) Flow cytometry analysis of EP cells (top) stained with either isotype control antibody: APC conjugate (gray histogram) or anti-CXCR4: APC conjugate (magenta histogram), and HP cells (bottom) stained with either isotype control antibody: APC conjugate (gray histogram) or anti-CD99: APC conjugate (cyan histogram). Histograms show a result representative from four biological replicates; mean values for percent positive are shown +standard deviation. (D) Western blot analysis of total protein extracted from UD, EP, CM, ECT, or HP cells and probed with antibodies directed against EOMES, SOX17, NKX2.5, AFP, and Tubulin; results shown are representative of at least two biological replicates. CM: cardiac mesoderm; DAPI: 4’,6-diamidino-2-phenylindole; EP: endoderm pro- genitor; HP: hepatic progenitor; UD: undifferentiated hESCs.

Article Snippet: The membrane was blocked with 5% milk in tris-buffered saline (TBS) at room temperature for 1 h, then primary antibodies mouse anti-EOMES (clone 644730/catalog # MAB6166, R&D Systems), goat anti-SOX17 (catalog # AF1924, R&D Systems), rabbit anti-NKX2.5, rabbit anti-AFP (catalog # SAB3500533, Sigma-Aldrich), and rabbit anti-Tubulin (catalog # 2148 S, Cell Signaling Technologies) were used to probe for the specified antigen overnight at 4 C. Membranes were washed three times in TBS with 0.1% Tween-20 (TBST) and probed with infrared dye–conjugated secondary antibodies, all from LI-COR (Lincoln, NE, USA): IRDye 800CW donkey anti-rabbit IgG (1:15,000 dilution; catalog # 926-32213), IRDye 680RD donkey anti-goat IgG (1:20,000 dilution; catalog # 926-68074), or IRDye 680RD donkey anti-mouse IgG (1:20,000 dilution; catalog # 926-68072), then washed three additional times with TBST.

Techniques: In Vitro, Quantitative RT-PCR, Standard Deviation, Immunofluorescence, Flow Cytometry, Staining, Control, Western Blot