What do cells look like after nuclear extraction swell/break step? - (Dec/14/2008 )

I've looked at millions of nuclear extraction protocols, but would like to use the hypotonic buffer, sell cells 15minutes, add np40 breaking cells, light spin for nuclear pellet. But it always says to check the cells to see if they have lysed. But what the heck am I looking for? I haven't been able to find any example pics out on the net. Could someone show me some, or tell me what I'm looking for.

Especially if I wanted to go for no detergent, just swell, and mechanially (dounce) disrupt, I really need to know what I am looking for.

Thanks

Hi

You can use trypan blue (or without) to look at the cell, as trypan blue will give better contrasting background. Before lysis you can see that the cells appear spherical and normal as what you would see under microscope. But after lysis, you will be able to detect lots of cellular debris, with a much smaller spherical structure, which is actually the nucleus, without the cytoplasm and cell membrane. If you have more of smaller spherical structure compared to larger ones, the better is your lysis.

...-...

Hmm..., I was just wondering how this works with brain tissue, especially neurons? When I dounce homogenize initially and then let the cells swell, does that actually work? Or does the homogenizing immediately break up the neuronal cells (since they are not really round)?

I was hoping someone would have or could point to some pictures before and after. But I guess I'll just have to osee for myself

Oh, my other thought was, what about the fact that I resect the brain tissue and immediately freeze it. Then later thaw and process it. The cells are still viable and will swell right? Yeah they should... right?

Hi,
Can't help you much with nuclear extraction from tissue, but I came across an interesting protocol which might be useful to you. You can watch it at http://www.jove.com/index/details.stp?ID=914.

Good luck