Journal: Bioconjugate Chemistry

Article Title: Enhancing Reactivity for Bioorthogonal Pretargeting by Unmasking Antibody-Conjugated trans-Cyclooctenes

doi: 10.1021/bc500605g

Figure Lengend Snippet: Figure 5. Translation of dual orthogonal coupling to different antibodies. (A) Anti-TfR and EpCAM antibodies had low TCO reactivity (12−15%) after direct amine-conjugation, but the PEG24− TCO increased reactive TCO density 3- to 4-fold. *MALDI-TOF analysis of the TCO−PEG24-modified anti-EpCAM antibody was limited by difficulties with desorption. (B) Flow cytometry results for TCO- and TCO−PEG24-modified anti-EpCAM and TfR antibodies on A431 cells after reaction with 10 nM tetrazine−QDs for 30 min, showing 5-fold signal enhancements for the PEG24−TCO. (C) Confocal images of A431 cells labeled with (i, iv) anti-EpCAM, (ii, v) anti-TfR, or (iii, vi) nonbinding control antibodies. The antibodies were modified with (i−iii) TCO or (iv−vi) PEG24−TCO at the highest densities and reacted with tetrazine−QD at 10 nM for 30 min. Scale bar: 20 μm. Error bars represent the standard error of at least three independent experiments.

Article Snippet: Monoclonal mouse antibodies specific for human EpCAM (IgG2b, clone 158206) and transferrin receptor (TfR, IgG1, clone 29806) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Conjugation Assay, Flow Cytometry, Labeling, Control

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Efficient generation of functional hepatocytes from human embryonic stem cells and induced pluripotent stem cells by HNF4α transduction.

doi: 10.1038/mt.2011.234

Figure Lengend Snippet: Figure 2 Hepatic differentiation of human ESCs and iPSCs transduced with three factors. (a) The procedure for differentiation of human ESCs and iPSCs into hepatocytes via DE cells and hepatoblasts is presented schematically. The hESF-DIF medium was supplemented with 10 μg/ml human recombinant insulin, 5 μg/ml human apotransferrin, 10 μmol/l 2-mercaptoethanol, 10 μmol/l ethanolamine, 10 μmol/l sodium selenite, and 0.5 mg/ml fatty-acid-free BSA. The L15 medium was supplemented with 8.3% tryptose phosphate broth, 8.3% FBS, 10 μmol/l hydrocortisone 21- hemisuccinate, 1 μmol/l insulin, and 25 mmol/l NaHCO3. (b) Sequential morphological changes (day 0–20) of human ESCs (H9) differentiated into hepatocytes via DE cells and hepatoblasts are shown. Red arrow shows the cells that have double nuclei. (c) The morphology of primary human hepatocytes is shown. Bar represents 50 μm. BSA, bovine serum albumin; DE, definitive endoderm; ESC, embryonic stem cell; iPSC, induced pluripotent stem cell.

Article Snippet: The differentiation protocol for the induction of DE cells, hepatoblasts, and hepatocytes was based on our previous report with some modifications.7 Briefly, in mesendoderm differentiation, human ESCs and iPSCs were dissociated into single cells and cultured for 3 days on Matrigel (Becton, Dickinson and Company, Tokyo, Japan) in hESF-DIF medium (Cell Science & Technology Institute) supplemented with 10 g/ ml human recombinant insulin, 5 g/ml human apotransferrin, 10 mol/l 2-mercaptoethanol, 10 mol/l ethanolamine, 10 mol/l sodium selenite, 0.5 mg/ml BSA, and 100 ng/ml Activin A (R&D Systems, Minneapolis, MN).

Techniques: Transduction, Recombinant