extranuclear Hoechst 33342 staining

Gary Warnes g.warnes at qmul.ac.uk
Thu Jun 7 04:07:32 EDT 2007

Dear David,

Previously I have found when labelling cells with Hoechst 33342 and
MitoTracker then homogenising the cells I have found that the mitochondria
not only have the MitoTracker but have a definite HO 33342 signal too which
I took to be the mtDNA, so I guess this constitutes some of the signal you
are seeing. Although I did fix the cells before homogenisation so the HO
33342 may have labelled the mtDNA because of the permeabilisation.

Gar Warnes

Head of Flow Cytometry & Imaging

Institute of Cell & Molecular Science

Barts & The London School of Medicine & Dentistry

University of London 



From: David.C.McFarland at gsk.com [mailto:David.C.McFarland at gsk.com] 
Sent: 05 June 2007 18:29
To: cyto-inbox
Subject: extranuclear Hoechst 33342 staining

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, 


David McFarland
Principal Scientist

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