Isolation of Mouse Primary Aortic Endothelial Cells by Selection with Specific Antibodies



Fig. 1
Steps in mouse aortic endothelial cell extraction and culture. (a) Dissection of the animal to expose the thoracic and abdominal cavities. (b) Isolation of the aorta and heart in the thoracic cavity. (c) Petri dish containing aorta and heart in saline solution or PBS. (d) Cleaned aorta. (e) Aortic strips in a Petri dish. (f) Aortic strips in Matrigel-coated plates. (g) Migration of primary cell from an aortic strip (outlined by the red dashed line) onto the surface of the culture plate. (h) Confluent EC monolayer




 


3.

Place the aortas in cold PBS or saline solution (see Note 3 and Fig. 1c).

 

4.

Under a stereomicroscope, use fine forceps to carefully remove all fat tissue from the aorta (see Note 4 and Fig. 1d).

 

5.

Dissect aortas longitudinally with a scalpel to obtain 0.5–0.8-mm-wide strips (see Fig. 1e).

 

6.

In sterile conditions (see Note 5 ) place the aortic strips lumen-face down in the wells of a 24-well culture plate coated with unset Matrigel. Incubate the plates in a humidified cell culture incubator at 37 °C until the Matrigel solidifies (at least 2 h) (see Notes 6 and 7 ).

 

7.

Cover the wells with 2 mL of EC medium and culture the aortic strips for a minimum of 1 week (see Note 8 and Fig. 1f). When cell colonies are formed (see Fig. 1g), remove the remaining aortic tissue from the well and collect the cells as follows:



  • Wash the attached cells with PBS at least three times.


  • Add trypsin and incubate at 37 °C to detach the cells (usually 5 min).


  • Block trypsin activity by adding EC medium supplemented with 10 % FBS, and pellet the cells by centrifugation at approximately 200 × g for 5 min.

 

8.

Resuspend the cells in EC medium containing 10 % of FBS and plate them in gelatin-coated plates (see Note 9 ). Incubate the cells to expand the culture (see Note 10 ).

 

9.

Select the ECs from the culture as follows:



  • To each well add anti-CD102 antibody (3.5 μL/mL EC medium) and incubate for 30 min at 4 °C in a shaker set at low velocity (see Notes 11 and 12 ).


  • Remove the medium containing the anti-CD102 antibody and wash the cells 2–3 times with PBS.

 

10.

To each well add EC medium containing an appropriate secondary antibody linked to magnetic beads (e.g., anti-rat IgG if using rat IgG anti-mouse CD102 as primary antibody). Secondary antibody should be diluted 3.5 μL/mL EC medium. Incubate for 30 min at 4 °C (see Note 13 ).

 

11.

Wash the cells with PBS (2–3 times) and detach them with trypsin (as described in step 7).

 

12.

Use a magnet to retrieve cells bound to magnetic beads.

 

13.

Wash the ECs retained on the magnet with PBS and culture them in EC medium in gelatin-coated plates (as described in step 8).

 

14.

Expand the EC culture as necessary (see Note 14 ).

 

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Sep 17, 2016 | Posted by in SUGERY, ORTHOPEDICS & ANESTHESIA | Comments Off on Isolation of Mouse Primary Aortic Endothelial Cells by Selection with Specific Antibodies

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