Wound scratch assay - (Sep/14/2016 )

Hi,

I'm new to this and have gotten a very vague protocol from someone in my lab, so would like to just get some clarification on some things.

The protocol says to coat a 6 well plate with collagen, but it says to coat with 100 uL of diluted collagen, and that seems very little to me and doesn't seem to be enough to cover the bottom of the well ( I haven't tried this out yet), but all the protocols I've seen online they put 1 - 2 mL diluted collagen per well. Which is correct?

Also the protocol says that you can make 4 scratches on the monolayer per well. All the videos/protocols I've seen only do one scratch per well. Does the number of scratches matter?

It's been a long while since I have done a scratch assay, but in general only one scratch is usually done per well. 100 ul can be enough to coat the bottom of a well of a 6 well plate, but it requires that you rock the plate fairly often to get it to do so. I would use a bigger volume. 

As you only have a vague protocol, I would do a search on Nature Protocols or Current Protocols and see if you can find a more specific protocol.