1. Isolate 10.5 - 12.5 dpc embryos from pregnant mouse by dissection and mince into 1-2 mm pieces with a scalpel. Suspend cells in 5ml PBS (w/o Mg++ or Ca++) on ice in a 50ml conical tube. Centrifuge at 1500rpm, 4¡ãC for 5 min. Remove supernatant by aspiration and resuspend pellet in 5ml PBS.

2. Add 5ml prewarmed PBS (Mg++, Ca++, Gibco BRL) containing 5 mg/ml collagenase (Worthington Biochemical Corporation) and 40 ¦Ìl of DNase I (stock; 30 U/¦Ìl, SIGMA). Shake at 37¡ãC for 45min, pipetting every 10min)

3. Pipette 40-50 times to separate cells into a single cell suspension. Add 10ml DMEM (Gibco BRL) containing 10 % FCS (Gibco BRL) and mix well.(to inactivate collagenase); Centrifuge at 1500 rpm, 4¡ãC for 5 min.

4. Remove supernatant by aspiration and resuspend cells in 10ml PBS. Filter cells through cell strainer (40 ¦Ìm mesh, FALCON), and Centrifuge, 1500 rpm, 4¡ãC for 5 min.

5. Resuspend Cells in 1-10 ml of DMEM/FCS (this volume depends on number of embryos).

6. Count cells under the microscope then transfer cell suspension (approximately 2x106 cells) into an Eppendorf tube. Centrifuge at 4000 rpm, 4¡ãC, 3 min.

7. Remove supernatant by aspiration and add 50¦Ìl of normal mouse serum (isolated from adult mice). Incubate on ice for 40 min. (blocking).

8. Add 1 ¦Ìl of anti-PECAM rat monoclonal antibody (0.5 mg/ml, PHARMINGEN) and mix gently. Incubate on ice for 45 min. (resuspending cells every 15min)

9. Wash with 900¦Ìl of PBS and pellet cells by centrifugation for a total of three times.

10. Resuspend in 50¦Ìl PBS and add 1¦Ìl secondary antibody, anti-rat donkey Cy3 conjugated antibody (Jackson Immuno Research). Incubate on ice for 20 min.

11. Wash with 900¦Ìl PBS and pellet by centrifugation three times.

12. After washing three times with PBS, the cells were sorted by FACScan (BECTON DICKINSON, San Jose, CA).

13. A 488nm laser was used to excite and both 530nm and 585nm collection filters were used to detect GFP and Cy3 positive cells, respectively.