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Endothelial wound healing (cell migration) assay

Posted on Friday, January 26, 2007

Description
This is a simple assay that can be used in any cell culture lab setup to test the effect of different compounds on endothelial cell migration.

Procedure
Grow endothelial cells in complete media.

Day 1: Trypsinize cells and add 4 x105 cells per well in a six well plate in complete media. After 8 h, change media to starvation media. Starve cells overnight in this media.

Day 2:
1) Remove media from cells.
2) Make a scratch wound across each well of the 6-well plate using a pipette tip. Make sure to use a very fine tip so that the cells come off the plate and the cell free area has sharp clear edges.
3) Wash three times with starvation media to remove any loosely held cells. Make sure that the wound area is free of cells.
4) Add starvation media containing required concentration of compound to be tested in 3 ml volume per well.
5) Take 0 hr. images.
6) Incubate at 37 degree C for 6 h. Take images.
7) Compare 0 and 6 hr images and calculate area of the wound closed using image J software.


Recipes


Supplies
Cell culture media
FBS (Invitrogen)
Complete media: Medium with FBS supplemented to 10%
Starvation media (SM): Medium with no FBS
Endothelial cells such as HUVEC, BAEC

75 cm2 vented flask (gelatin coated) (Biocoat)
gelatin coated 6-well plate (Biocoat)


Tips
1) The media used is the specific media for that cell type.
2) One can also try using media containing low percentage FBS to obtain faster closing of the wound.
3) The time of incubation can be increased or decreased based on the type of cells used (how fast they migrate) and the compound being tested.


Submitted by: sci2k1

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