Hi peers,

I have been optimizing a dextran experiment in my laboratory by which I want to permeabilize the plasma membrane but not the nuclear one. That is, because I want to assess the transport of dextran molecules from the exterior (cytosol/Extracellular environment) into the nucleus.

Like I said I have been trying to optimize the protocol, trying different incubation times with digitonin and concentrations too. The closer that I have gotten to get this partial permeabilization (understood as PM permeabilization but not disruption of the NM) of cells is incubating cells for around 7 min with a concentration of 30 ug/mL of digitonin (after titration). However, I have noticed that some cells, no matter what, remain not being permeabilized. I have adjusted and played with the conditions mentioned above quite a lot and to me it looks like a sharp change. That is, incubating cells for 7 min/30 ug.ml of digitonin results in around 60% of cells being properly permeabilized, but if I try to increase this proportion it results in all if not all cells permeabilized. I am afraid that this point may be past what I am looking for and I was wondering if anyone has encountered this heterogeneity in the permeabilization of cells using digitonin. Thank you very much in advance.

Additional info on the experimental setup: I am using Mouse Embryonic Fibroblasts and I perform the experiment in Transport buffer, described here: Article Clathrin inhibitor Pitstop-2 disrupts the nuclear pore compl...