Dear Ning Xu, apologize if I am not of help but nevertheless I would like to add here the URL for the other thread you "are coming from" with your unfortunately hitherto unanswered question there: https://www.researchgate.net/post/Scratch_Assays_with_keratinocytes?exp_tc=tprc

Best wishes and good luck,

Hello Ning Xu,

I have also been working with keratinocytes (not primary cells but E6/E7 immortalized cell lines) that showed a high migratory potential in scratch assays. I then used the BD BioCoat Matrigel invasion chambers (BD Biosciences Discovery Labware, #354480) to comfirm a higher invasive phenotype of these cell lines. Matrigel chambers also contain a membrane with 8µm pores, but the membrane is covered with Matrigel. Matrigel is composed of extra cellular matrix components. In order to migrate through the pores, the cells must digest away the Matrigel layer. The better they digest it away, the more invasive they are. In my case, the keratinocytes express high concentrations of kallikrein-related peptidases and matrix metalloproteinases. These enzymes are responsible for Matrigel degradation. In invasion chambers without Matrigel (used as the control for calculation of invasiveness), these keratinocytes are not higher invasive compared to wild-type keratinocytes. So if your cells are not migrating through a usual 8µm pore membrane, they maybe show a higher invasiveness in Matrigel assays.

But it's a little bit expensive! A set of 24 chambers costs about € 400,- and the control chambers cost about € 200,-

Best regards!

Hi Ning

In Boyden chamber assay, keratinocytes migrate well through a pre-coated PET membrane by fibronectin or collagen or vitronectin...Was your membrane coated?

Francois

Dear all,

Thanks a lot for your replies :-)

Francois, how do you coat the PET membrane, for example by collagen? Will this coating 'close' the pores on PET membrane?

I tried to detect the effect of a microRNA in human primary keratinocyte migration. By Boden chamber assay (uncoated PET membrane, attract cells with growth factor supplement+10% FCS in outer chamber, 24 hour migration), I found this miRNA increases cell migration. However, by scratch wound healing assay, this miRNA does not affect cell migration. Do you think how should I interpret these data? Which assay is more relevant to skin wound healing?

Ning

Hope you have figured out the best answer.

Per my experience, Scratch assay does better job than other approaches to study wound healing in vitro/in culture.

Also, make sure you remove the cells inside of the chamber completely when you use the Boyden chamber assays, and the time to allow cell migrating through the chamber is a key point to produce meaningful result

You should also consider the effect of keratinocyte proliferation in the scratch wound assay. If your miRNA is affecting the proliferation as well as migration, it could confound the results. You can inhibit proliferation by treating the cells with mitomycin C before scratch wounding.