(A) Flow cytometry analysis of C57BL/6 mouse splenocytes processed with EasySep™ Mouse CD11c Positive Selection Kit II and labeled with Anti-Mouse CD11c Antibody, Clone N418, Alexa Fluor® 488. Histograms show labeling of splenocytes (Start) and isolated cells (Isolated). Labeling of the start cells with an Armenian hamster IgG Alexa Fluor® 488 isotype control antibody is shown in the bottom panel (solid line histogram). (B) Flow cytometry analysis of C57BL/6 mouse splenocytes processed with EasySep™ Mouse CD11c Positive Selection Kit II and labeled with Anti-Mouse CD11c Antibody, Clone N418, Alexa Fluor® 488 and an anti-mouse CD317 antibody, APC. (C) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with Anti-Mouse CD11c Antibody, Clone N418, Alexa Fluor® 488 and an anti-mouse MHC class II antibody, APC. (D) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with an Armenian hamster IgG Alexa Fluor® 488 isotype control antibody and an anti-mouse MHC class II antibody, APC.
Figure 2. Data for PE-Conjugated
(A) Flow cytometry analysis of C57BL/6 mouse splenocytes processed with EasySep™ Mouse CD11c Positive Selection Kit II and labeled with Anti-Mouse CD11c Antibody, Clone N418, PE. Histograms show labeling of splenocytes (Start) and isolated cells (Isolated). Labeling of the start cells with an Armenian hamster IgG PE isotype control antibody is shown in the bottom panel (solid line histogram). (B) Flow cytometry analysis of C57BL/6 mouse splenocytes processed with EasySep™ Mouse CD11c Positive Selection Kit II and labeled with Anti-Mouse CD11c Antibody, Clone N418, PE and an anti-mouse CD317 antibody, APC. (C) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with Anti-Mouse CD11c Antibody, Clone N418, PE and an anti-mouse MHC class II antibody, APC. (D) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with an Armenian hamster IgG PE isotype control antibody and an anti-mouse MHC class II antibody, APC.
Figure 3. Data for Unconjugated
(A) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with Anti-Mouse CD11c Antibody, Clone N418, followed by anti-hamster (Armenian) IgG, FITC and anti-mouse MHC class II, APC. (B) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with a hamster (Armenian) IgG, isotype control antibody followed by anti-hamster (Armenian) IgG, FITC and anti-mouse MHC class II, APC.
Figure 4. Data for APC-Conjugated
(A) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with Anti-Mouse CD11c Antibody, Clone N418, APC and Anti-Mouse CD45 Antibody, Clone 30-F11, Alexa Fluor® 488 (Catalog #60030AD). (B) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with an Armenian hamster IgG APC isotype control antibody and Anti-Mouse CD45 Antibody, Clone 30-F11, Alexa Fluor® 488. (C) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with Anti-Mouse CD11c Antibody, Clone N418, APC and an anti-mouse MHC class II antibody, FITC. (D) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with an Armenian hamster IgG APC isotype control antibody and an anti-mouse MHC class II antibody, FITC.
Figure 5. Data for Biotin-Conjugated
(A) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with Anti-Mouse CD11c Antibody, Clone N418, Biotin followed by streptavidin (SAV) APC and Anti-Mouse CD45 Antibody, Clone 30-F11, FITC (Catalog #60030FI). (B) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with a biotinylated Armenian hamster IgG, isotype control antibody followed by SAV APC and Anti-Mouse CD45 Antibody, Clone 30-F11, FITC. (C) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with Anti-Mouse CD11c Antibody, Clone N418, Biotin, followed by SAV APC and an anti-mouse MHC class II antibody, FITC. (D) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with a biotinylated Armenian hamster IgG, isotype control antibody followed by SAV APC and an anti-mouse MHC class II antibody, FITC.
Figure 6. Data for FITC-Conjugated
(A) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with Anti-Mouse CD11c Antibody, Clone N418, FITC and Anti-Mouse CD45 Antibody, Clone 30-F11, APC (Catalog #60030AZ). (B) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with an Armenian hamster IgG FITC isotype control antibody and Anti-Mouse CD45 Antibody, Clone 30-F11, APC.
Figure 7. Data for PerCP-Cy55-Conjugated
(A) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with Anti-Mouse CD11c Antibody, Clone N418, PerCP-Cy5.5 and an anti-mouse CD317 antibody, APC. (B) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with an Armenian hamster IgG PerCP-Cy5.5 isotype control antibody and an anti-mouse CD317 antibody, APC. (C) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with Anti-Mouse CD11c Antibody, Clone N418, PerCP-Cy5.5 and an anti-mouse MHC class II antibody, FITC. (D) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with an Armenian hamster IgG PerCP-Cy5.5 isotype control antibody and an anti-mouse MHC class II antibody, FITC.
Figure 8. Data for PB-Conjugated
(A) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with Anti-Mouse CD11c Antibody, Clone N418, Pacific Blue™ and Anti-Mouse CD45 Antibody, Clone 30-F11, FITC (Catalog #60030FI). (B) Flow cytometry analysis of C57BL/6 mouse splenocytes labeled with an Armenian hamster IgG Pacific Blue™ isotype control antibody and Anti-Mouse CD45 Antibody, Clone 30-F11, FITC.
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Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering. Haraguchi Y et al. Journal of Tissue Engineering and Regenerative Medicine 2015 DEC
Abstract
In this study, a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension, only a few aggregated cells were observed. However, after 3 days, culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry, immunocytochemistry and quantitative RT-PCR, and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium, expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore, the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A, BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes, including HCN4, MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes, including pacemakers. Moreover, when cardiac cell sheets were fabricated using differentiated cardiomyocytes, they beat spontaneously and synchronously, indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering.
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Legal Statement: Alexa Fluor and Pacific Blue are trademarks of Life Technologies Corporation. Antibodies conjugated to Alexa Fluor® or Pacific Blue™ are licensed for internal research use only and sale is expressly conditioned on the buyer not using the antibody for manufacturing, performing a service or medical test, or otherwise generating revenue. For use other than research, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com. PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED. FOR ADDITIONAL INFORMATION ON QUALITY AT STEMCELL, REFER TO WWW.STEMCELL.COM/COMPLIANCE.