13. Wash the cells twice for 5 min in PBS.

14. Put one drop of mounting medium, add coverslip and seal with

nail polish.

15. Observe the slides and acquire the image under the Confocal

microscope.

3.5.2

Flow Cytometry

At the culture of day 5 and 8, the differentiated cells in flasks under

different culture conditions are harvested and prepared for

immunostaining.

1. Dissociate the cells into single cells with 0.05% trypsin and

0.1% EDTA.

2. Add trypsin inhibitor solution and centrifuge for 5 min,

500  g at 4
C and discard the supernatant.

3. Wash cells with cell washing buffer (PBS containing 5% BSA).

Count the cells and resuspend in 5% BSA of PBS at

1  10⁶ cells/mL.

4. Transfer 50 μL cell suspension to test tubes.

5. Add the diluted fluorescent conjugated, purified primary anti-

bodies (CD31, CD34, and CD43) into test tubes at dilutions

recommended by the manufacturer. Controls include: negative

(no stain added), isotype control (with similarly labeled, non-

specific primary antibody).

6. Incubate on ice for 30 min in the dark.

7. Wash each sample three times with 2 mL cell washing buffer,

spin down at 500  g for 5 min at 4
C.

8. Add 500 μL PBS to each pellet and resuspend cell sample.

9. Example the cell samples using FACS Calibur flow cytometer

(BD).

10. Analyze the data with FlowJo software, version 10.0.7.

4

Notes

1. If not used immediately, the coated culture plate must be sealed

with parafilm to prevent evaporation of the Matrigel^® solution

and the plates can be stored at 2–8
C for up to 1 week after

coating.

2. When cell dissociation about 2 min in the incubator, you

should check the cells state to make sure the colonies are

loose and fall off from the bottom of the dish or flask. Do not

overdigest the cells to generate a single-cell suspension.

3. Gently resuspended the cell pellet with a pipet to avoid gen-

erating single-cell suspension. Cell aggregate size can be

64

Xiaohua Lei et al.