9. Incubate the cell in primary antibody solution for overnight at

4
C (or 1 h at room temperature with fluorescent-Dye conju-

gated antibodies).

10. Wash the cells three times with PBS in order to reduce the

fluorescent background.

11. Prepare the mixed solution containing the second antibody

(1:200 dilutions) and Hoechst 33342 (0.1–1 μg/mL) in 5%

donkey serum PBS.

12. Incubate the cells in mixed solution for 1 h at room

temperature.

Fig. 4 Hematopoietic progenitor cells induction from human embryonic stem cells (hESCs) in RPM. Bright-field

images of representative cellular morphology of hematopoietic progenitor cells with grape-like clusters (a) and

with floating round cells at culture of day 8 (b). Scale bars, 50 μm. (c) Representative immunostaining images

of differentiating cells in RPM at day 8 for CD34 and CD43. Scale bars, 50 μm. (d) Representative flow

cytometry results of surface markers CD34 and CD43 at day 9 from normal gravity (NG) and simulated

microgravity (SMG)

Hematopoietic Stem/Progenitor Cell Differentiation in Random Positioning. . .

63