extranuclear Hoechst 33342 staining

David.C.McFarland@GSK.COM David.C.McFarland at GSK.COM
Tue Jun 5 13:28:59 EDT 2007


Hello all,

I have an interesting finding and was wondering if anyone else out there 
has seen something similar.  I've identified & sorted a population of 
cells from rat lysed whole blood that displayed Hoechst (HO) 33342 
staining intensity (350 nm ex, 530 nm em) about 10X that of surrounding 
leukocytes.  In addition, the forward & side scatter signals were well 
below lymphocytes.  Fluorescence imaging revealed that sorted leukocytes 
had expected nuclear staining.	The 10X HO population had clear nuclear 
staining, but also a fair amount of diffuse cytosolic staining.  They were 
also similar in size (~10 um).	No apoptotic or mitotic features were 
readily apparent.  Can someone describe why I would see this type of 
staining pattern in the cytosol & exactly WHAT is staining?  Is there a 
cellular pathology or biological phenomenon that would explain this?  Is 
it possible that I am visualizing non-genomic DNA staining such as dsRNA, 
mtDNA or other?  I should point out that these animals were drug treated, 
so there is an associated pathology with several organs.  The drug is not 
expected to be fluorescent or DNA-binding.  Also, blood samples were 
frozen at -70C before thawing, staining & sorting.

Thanks in advance,

Dave

David McFarland
Principal Scientist
GlaxoSmithKline
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