[박상규] Transwell migration of endothelial cells

[Anonymous]

Transwell migration of endothelial cells

 

 

** must be prepared before experiment**

 

1.     0.5mg/ml gelatin in 1XPBS=> autoclave or 0.2 um filtering. 물은 3차 증류수

2.     8um pore size transwell chamber (Costar)

3.     Positive control : ex) VEGF

4.     cotton swab

5.     methanol (100%), hematoxylin, 1X PBS.

 

 

1.     Coat the out-side membrane of chamber with 10ul 0.5mg/ml gelatin and spread to cover whole area of membrane.

2.     Stand the transwell on clean bench under U.V till dry

3.     Fill the lower chamber with 300ul serum free media containing VEGF (0.7nM) or your interesting molecule.

4.     Resuspend the BAECs with serum free media and load 250ul (2-5X10⁴ cells) volume to upper chamber and incubate for 3-10 hrs in CO2 incubator.

5.     To visualize the migrated cell, remove the media and fix with 100% methanol for 10 min. and stain with 1/2 diluted hematoxylin for 1 hrs.

6.     Wash the upper chamber with 1X PBS 4 times and, carefully and completely remove the BAECs of the upper side membrane with cotton swab ( Be careful not to scratch the membrane). ç This step is really important

7.     Carefully cut the membrane with razor blade and set the membrane onto slide glass. ç This step is really important

8.     Mount with 80ul mounting solution (Gel/mount; Biomedia) and dry for at least 1 hrs.

9.     observe the migrated cells with 20X light microscope.

10.  Count the cells at least 5 different areas and make mean value.

 

*** And also, Do Not discard the lower chamber because there could be migrated cells. So, follow the above protocol to stain the cells and count the migrated cells.

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