intracelluar staining without killing cells - (Nov/18/2010 )
Hi, folks,
Do you know how to do an intracellular staining without killing the cells? The classical way to fix/perm the cells will definitely kill them.
Thanks.
M
It all depends on what you're trying to see. I'm not sure about "staining" but the way most people would do live cell imaging would be to transiently transfect the cells with a plasmid that expresses a fluorescently labeled protein, however this is going to depend on overexpression. If you're trying to see certain organelles, invitrogen has fluorescent dyes that will localize to lysosomes, mitochondria, etc. Unless I'm mistaken, I think you're going to have to rely on dyes though because I don't think you'll be able to get an antibody to cross the cell membrane while it's still alive.
My purpose is to stain intracellular proteins with fluorescence-labeled antibodies and then sort positive cells for RNA extraction. The current protocol is to perm and fix the cells and then sort. Researchers had done this before and isolated RNA quite successfully from fixed cells. However, I'm still concern about the quality of mRNA, since I will perform single cell RNA isolation. IF ever possible, I will try to keep the cell alive.
kfunk106 on Thu Nov 18 20:44:56 2010 said:
It all depends on what you're trying to see. I'm not sure about "staining" but the way most people would do live cell imaging would be to transiently transfect the cells with a plasmid that expresses a fluorescently labeled protein, however this is going to depend on overexpression. If you're trying to see certain organelles, invitrogen has fluorescent dyes that will localize to lysosomes, mitochondria, etc. Unless I'm mistaken, I think you're going to have to rely on dyes though because I don't think you'll be able to get an antibody to cross the cell membrane while it's still alive.
Unfortunately there is no way to stain intracellular proteins by FL-labelled antibodies without fixation and permeabilization.
Antibodies are too big...For DNA or RNA analysis after sorting the fix/perm procedure is ok.
The only way is to try to find a surface marker (if any...) suitable for staining and for the determination of your cells of interest