I'm having a lot of trouble trying to do the scratch, because patches of cells come out and then I don't have a perfect line for the assay. I used a p200 tip and washed with PBS after the scratch and before treating the cells with my drug test. Any suggestions?
I have found it better to wash cells once with either PBS or serum free media, then lightly scratch the cells with a p10 tip in 1 fluid motion (like writing with a pen not cutting with a scalpel). Wash the cells twice with either PBS or serum free media to remove detached cells, then replace media with treatment media.
You should scratch the cells once they become confluent to avoid clumping or group shedding/tearing and avoid swirling the media too much with caco2 cells as they can stick too each other stronger than they stick to the plate, which can cause the edge to lift or tear in places
A few things you may try.
1. scratch in a swift and quick stroke, instead of dragging the tips slowly which may tear the monolayer
2. Test different timepoints after the cells reaching confluence. It is good to have a confluent monolayer, but not until the cells attach to each other too tightly.
3. If possible, try other cell lines. Caco-2 tend to have very "tight" connections.
the most important thing while making a scratch is that it should happen in "one go" fast and precise. You can do it with a sterile tooth pick, if you have problems with a p200 tip.I would suggest, remove the entire media, make a brisk confident scratch and wash with PBS and finally add fresh media.
Best of luck
I have found it better to wash cells once with either PBS or serum free media, then lightly scratch the cells with a p10 tip in 1 fluid motion (like writing with a pen not cutting with a scalpel). Wash the cells twice with either PBS or serum free media to remove detached cells, then replace media with treatment media.
You should scratch the cells once they become confluent to avoid clumping or group shedding/tearing and avoid swirling the media too much with caco2 cells as they can stick too each other stronger than they stick to the plate, which can cause the edge to lift or tear in places
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