BD Pharmingen™ Purified Mouse anti-Human Sox17

Immunofluorescent staining of Sox17 in definitive endoderm derived from human embryonic stem (ES) cells. H9 human ES cells (WiCell, Madison, WI) passage 35 grown on an irradiated mouse embryonic feeder layer were differentiated to definitive endoderm for 3 days (D'Amour et al, 2005) in RPMI medium supplemented with 0.5% FBS, 1× L-glutamine, and 100 ng/ml Activin A (R&D Systems). The cells were fixed with BD Cytofix buffer (Cat. No. 554655), permeabilized with 0.1% Triton™ X-100, and stained with Purified Mouse anti-Human Sox17 monoclonal antibody (pseudo-colored green) at 1.2 µg/mL. The second-step reagent was Alexa Fluor® 488 goat anti-mouse Ig (Life Technologies), and counter staining was with Hoechst 33342 (pseudo-colored blue). The image was captured on a BD Pathway™ 435 Cell Analyzer and merged using BD Attovision™ Software.

Flow cytometric analysis of Sox17 in definitive endoderm derived from human embryonic stem (ES) cells. H9 human ES cells (WiCell, Madison, WI) grown on an irradiated mouse embryonic feeder layer were differentiated to definitive endoderm for 3 days (D'Amour et al, 2005) in RPMI medium supplemented with 0.5% FBS, 1× L-glutamine, and 100 ng/ml Activin A (R&D Systems). Day-3 differentiated cells were fixed with BD Cytofix buffer (Cat. No. 554655) and permeabilized with BD™ Phosflow Perm buffer III (Cat. No. 558050). The cells were stained with either Purified Mouse IgG1, κ isotype control (dashed line, Cat. No.555746) or Purified Mouse Anti-human Sox17 antibody (solid line) at matched concentrations. The second-step reagent was APC goat anti-mouse Ig (Cat. No. 550826). The histograms were derived from gated events based on light scattering characteristics of the H9-derived endoderm cells. Flow cytometry was performed on a BD LSR™ II flow cytometry system.