Hello,

We've been trying to perform soft agar assays (for the first time) over the past month. We do them in 6-well dishes. Everything seems to be going fine except that some of the agar discs are coming loose over time (3-4 days) and they begin to float around in the media, which makes it difficult to change the media. Does anyone know why this happens, and what we can do to prevent it?

Thanks...

Hi! What conc/depth of overlay are you using?
In the meantime, here are a couple of quick and fairly trivial suggestions:
Use 6 well plates instead of 6 cm dishes - even if its the same surface coat/material, some how the plates are more stable when you transfer them from hood to incubator - without shake!
Also, wait a fews mins longer in the room temp gelling step before overlaying, and overlay very slowly along one edge only.

Thanks samm for the reply...

We've been using 1.5 ml of 0.5% (final working concentration) agar for the bottom layer, and 1.5 ml of 0.35% for the top. We have been using 6-well dishes, and we wait for 20 minutes before puring the top layer - but we haven't been too particular as to how we pour the gels, so that might be the answer.

Thanks again - We'll give it another shot.

Hi! Did that work out for you? Also, check out the new defined matrices that are now available from many vendors: you can even set up combination plates and do away with MatriGel entirely, though the soft agar assays will almost certainly still be around!

I also do my soft agar assays in 6-well. I pour 1.5 ml of 0.7% (final) per well and let it sit on my bench for about 30 minutes before preparing my cells for the top layer. I also pre-warm my plate (with the bottom layer) at 37 degrees while I'm counting my cells right before plating them. I plate in 1 ml 0f 0.35%, and again let it sit on my bench for about 30 minutes before adding media. Hope this helps!

Thanks for all the suggestions. We've re-tried it a few times now and it looks like the secret is to give plenty of time to allow the top agar to solidify, like you suggested. Things are working better now.

I've peformed many soft agarose assays for hematopoietic cells using 0.36% low-melting point agarose. We always refrigerate the plates for about 5-10 minutes before putting them in the incubator to make sure they solidfy.

Here are my questions about Soft Agar assays.

How firm should the top layer be when you put it back in the incubator? Solid like agar plates, liquid, gelatinous? And how can you tell if it is juuuust right?

What if you put it back while it is still liquid? Will it ever solidify?

Ed