How to make Matrigel layer as thin as humanly possible? - Our technique relies on a VERY close contact of cell with coverslip (Feb/15/2008 )

Hi there
I'm a new post-doc working on a project which uses a technique similar to TIRF/TIRM to image cells. This technique requires cells to ideally be within 50nm of the coverslip surface(!)
I want to use Matrigel (or something similar) to culture my cells - primary rat embryonic hippocampal neurons - since this seems better for their survival, neurite outgrowth, etc (at least from what I see in the literature..)
I wondered if anyone has experience REALLY thinning this stuff out so that it can form as thin a layer as possible, since most protocols I see result in quite a thick layer of the stuff..

Any help appreciated, new member
Hi!

Cheers,
Kev

dilute Matrigel to homeopathic dosis and start coating

Hi,

We use dilluted Matrigel for coating plates, the layer is pretty thin, and the cells develop well (primary muscle cells in my case). I used to stain the cells with fluorescence and there was no problems.


Here is the protocol we use :

Coat 4-well slides with 10% Matrigel

Several hours before plating cells,
Thaw Matrigel aliquot on ice (10% Matrigel in DME medium)
Dispense 500 uL Matrigel into each well
Ensure that wells are fully coated by shaking plate, then tilt and remove the Matrigel.
Dry the plate at RT until you plate the cells.

You have to make sure you remove a much matrigel as you can and perform all the steps on ice! If you coat the plates at RT, the matrigel will solidify very fast and you will not get a thin layer.

Hope that help you!
Fabien

We dilute matrigel 1:40 in DMEM and use around 150ul/ well in a 24 well plate or 500ul/ well in a 6 well plate.
Add the matrigel and spread it around the well and then turn it towards you and remove the matrigel. Let the plate dry (do not cover) for 30-60 min and it is ready to use and evenly coated.

Thanks guys, that's cool to have a range of ideas. I'll start pretty dilute I think, maybe somewhere just above homeopathic
Just out of interest, do you know if iits possible to label the Matrigel so you can titrate the layer thickness somehow?

Cheers,
Kevin