Nuclear isolation techniques
kschafer@lucy.tli.org
Thu, 15 Aug 1996 12:24:21 +0000
Dear Flow-ers,
I have been attempting to look at cell cycle-dependent
subcellular localization of a protein. I have been staining
fixed cells with PI and FITC and gating and sorting the cells
into G0-G1-S and G2-M populations. After sorting I have been
examining the cells with fluorescence microscopy to see if the
FITC is located in the cytoplasm and/or nucleus. I have been
unable to easily distinguish nuclear staining from staining in
the overlying cytoplasm. Unfortunately, I don't have access to
confocal microscopy. Alternatively, I have tried to isolate
nuclei (i.e. strip off the cytoplasm), stain them, and then sort
them. However, after all is said and done, the nuclei are
either: so sticky they won't go through the flow, or I have just
debris, or the cells still have the majority of the cytoplasm
still in place. Does anyone know of a method for isolating
nuclei, antibody-FITC staining them, and then sorting them?
Should I stain whole cells and then isolate nuclei (strip off
cytoplasm)? Am I instead going about this all wrong? Might there
be a better way to sort cells into G1-S and G2-M and then look at
the localization of a protein?
Thanks in advance for any input.
Ken Schafer, DVM
kschafer@lucy.tli.org
Lovelace Inhalation Toxicology Research Institute
Albuquerque, NM
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