Sorry I have been slow in replying. Unusually there hasn't been
much response to my question about BrDU methods but looking nearer to
home I've come across three variations which basically differ in the
way the DNA is denatured prior to staining with antibody.
1. You can use acid/alkali conditions
2. You can heat the cells to 90C
3. You can treat the cells with DNAse I
Unfortunately, none of my collegues have experience in comparing
these methods. However, my bias is towards the DNAse method because
my guess is that this will give better recovery of the fixed cells.
All the methods seem to work well for the people concerned. One other
method which has already been on the bulletin board is attached
below. I would still like to hear from anyone that has compared
these methods or can suggest better ones
==================================
Date sent: Tue, 25 Jul 1995 09:03:09 -0500
From: ZBIGNIEW DARZYNKIEWICZ <darzynk@nymc.edu>
To: cyto-inbox
Subject: Re BU-1 clone
To the ongoing discussion on a possibility of simultaneous analysis
of BrdUrd incorporation and cell immunophenotype let me add the
following suggestion: the SBIP method of analysis of BrdUrd
incorporation (See June issue of Cytometry and Cancer Res., 54:4289,
1994) does not require DNA denaturation and is compatible with
surface immunophenotyping. Brief fixation with formaldehyde
stabilizes e.g. FITC labeled antibody, the cells are then briefly
illuminated with UV light, permeabilized and DNA strand breaks
labeled with digoxygenin or biotin conjugated dUTP. The later are
detected with antibody or avidin conjugated fluorochrome of another
color than FITC. Cell morphology is well preserved.
Zbigniew Darzynkiewicz
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From: Dr Richard Darley
LRF Preleukaemia Unit
Department of Haematology
UWCM
Cardiff, CF4 4XN
U.K.
tel (+44) 12 22 74 45 24
Fax (+44) 12 22 74 45 23
e-mail: darley@cardiff.ac.uk