In short, I would like to let the cytoplasm "leak" out of adherent cells and retain just the membrane and other cellular compartments. I tried a hypotonic buffer with 10 mM Hepes, 2 mM EGTA and 2 mM MgCl2. It worked partly, but depending on the cell density, some cells remained intact. I also tried hydrodynamic force, but most cells detached from the surface. Does anyone have better ideas to selectively remove the cytoplasm and have the rest of it stuck for immunofluorescence?
My collague
Dr, Haim Weinhouse
developed methods for single-celled, and if I recall, membrane stabilizers w employed i addition to the perforation stage. The work was directly to enhancing permeability for small, charged structures.
Sinecerly and best of luck !
-Pete
Re: Dr. Haim Weinhouse (ret.)
May be you can increase the volume of hypotonic solution you use, for dense cultures. At low volumes, the cytoplasm that come out early might alter hypotonic solution to isotonic one. By increasing the volume, this can be reduced. But any such osmotic shock will result in pyknosis and subsequent swelling but still attached to substratum by a thin thread like stalk. You might handle the cells very gently during subsequent immunofluorescence procedure. Good luck...!!
Hi Goodwin,
Valid point indeed. That could probably be the reason why I see partial lysis in hypotonic solution. I was worried about diluting the sample too much by using a larger volume. But I will give it a try anyway and see if I can get complete lysis.
Thanks,
Pradeep
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