Listed below are all of the replies to my question about Hoechst uptake
in apoptosis. I have done a minimum of editing. I have also included
the addresses of some web pages that I found helpful.
http://nucleus.immunol.washington.edu/Research_facilities/Apps/apoptosis.html
http://nucleus.immunol.washington.edu/Research_facilities/Refs/schmid_apoptosis.html
http://www.icnet.uk/axp/facs/davies/apop.html#HPI
http://www.cyto.purdue.edu/hmarchive/Feb94-May95/0664.html
http://www.biotech.iastate.edu/facilities/CELLHYB/apop.htm
Date: Fri, 29 Aug 1997 17:36:30 -0700
From: Tom_Frey@bdis.com (Tom Frey)
I have used H33342 with thymocytes with good result. I used 1 ug/mL for
10
minutes. Since this is a kinetic effect you must pay attention to time
and
concentration. I used linear amplification of the Hoechst. See
Cytometry
21:265.
I have never gotten this method to work with HL-60 treated with
camptothecin. I
personally believe that this is on of the least reliable apoptosis
methods. I
am frankly surprised that 33258 shows discrimination in any system, it
crosses
membranes much less well than 33342. Note, however, that PI and other
dyes that
don't cross intact membranes (I would include 33258 in this class) do
often
stain apoptotic cells better. Again use linear. This method works more
often
than the kinetic 33342 method but is still quite unreliable in my
experience.
For some further info on what has worked for me see the July Cytometry
28:253.
_______________________________________________________________________________
From: "Mark A. Miller" <mamiller@biochem.dental.upenn.edu>
Date: 8/29/97 11:16 AM
Who has expeience using Hoechst uptake as an indicator of apoptosis?
1) Are you using 33342 or 33258? Concentration?
2) Are you using log or lin acquisition for the Hoechst?
3) Do you use PI exclusion? How about 7-AAD? Concentration?
4) How long do you expose the cells to the dyes before reading? Temp?
My concerns mostly center around the third question... the 7-AAD signal
just doen't look like I expect it should. Several labs I work with have
been using the following protocol with various cell types, including
chondrocytes, HL-60, and fresh human T cells:
1.Resuspend at least 0.5 x 10^6 cells in 12x75mm tubes at a
concentration of 10^6 cells/ml.
2.Add Ho 33258 at a final concentration of 1ug/ml to samples.
3.Incubate the samples in a 37 C waterbath for 7 minutes.
4.Remove samples from the waterbath and immediately place on ice.
5.Add 7-AAD at a final concentration of 1ug/ml to samples.
6.Incubate on ice for 10 minutes.
7.Analyze samples, with log amplification for both Hoechst and 7-AAD.
_______________________________________________________________________________
Date: Mon, 1 Sep 1997 00:33:48 +1000
From: Bob Ashcroft <cytomat@netcore.com.au>
It's been years...
My experience is to use 1 uM for 2 minutes to get , live, apoptotic and
dead.
_______________________________________________________________________________
Subject: Re: Question: Hoechst uptake in apoptotic cells
Date: Sun, 31 Aug 1997 19:36:27 -0400 (EDT)
From: Howard Shapiro <hms@shapirolab.com>
Hoechst 33258 and 33342 have very different permeability properties;
33342
will get across the intact cytoplasmic membranes of most mammalian
cells,
while 33258 is typically excluded. If you are working with fixed or
permeabilized cells, or with chromosome preps, the two dyes can be used
more
or less interchangeably; for apoptosis assays, which depend critically
on
membrane permeability changes, substituting one dye for another is
asking
for trouble. I also get the impression that if one or two of the
published
apoptosis assays worked uniformly reliably and well with a broad range
of
cell types, we wouldn't see new apoptosis assays published every other
week.
______________________________________________________________________________
Date: Tue, 02 Sep 1997 10:26:45
From: "Dennis J. Young" <djyoung@ucsd.edu>
We tried a couple times and gave up. We may try again.
According to the protocols in Flow Cytometry (and others, Schmid,
Ingrid, I
think), you need to wash the HO342. It's the difference in the rate of
accumulation, not +/-. Also, have you tried PI. You can use single UV
laser. I know, you want to use surface markers, but this way you can
optimize the HO342 step.
(LOG acquisition for Hoechst)
______________________________________________________________________________
Date: Tue, 02 Sep 1997 11:09:40 -0400
From: Louis King <kingl@pilot.msu.edu>
Check the message from Dr Shapiro as he is right on. You must use HO
342 &
be aware that it stains DNA but you are in fact using its detection of
the
MDR transport pump. This lab looked at HO342 vs PI detection (as plasma
membrane integrety dye) and published a labelling protocol for detection
of
viable cells in J Immunol. Methods. 171:1-16 1994. Ho342 labelling was
done for 15 min at 1 ug/ml at 37C. In fact I will suggest trying lower
HO342 concentrations and shorter incubation times. 7AAD has always
given
evidence of PE fluorescence quenching in this lab & thus we do not use
it.
Thus in the published protocol both PI (1ug/ml & 3 min before FACS) and
HO342 fluorescence data are taken on log scale. Examinination of HO
33342
literature shows use of concentrations up to 10 ug or greater for
detection
of DNA cell cycle. I think you will not be happy with detection of
Apopotic cells from cell cycle staining. The method in the above
reference
continues to detect the earliest apoptotic cells including those showing
Annexin V binding from its first appearance. If I can be of more
help--email.
______________________________________________________________________________
Date: Tue, 2 Sep 1997 16:26:57 +0100 (BST)
From: Derek Davies <daviesd2@icrf.icnet.uk>
Yes, it was [log acquisition], sometimes the mean value of the apoptotic
population can be
20+ times that of the live cells.
I use 1ug/ml for 5 minutes at room temperature. This, I have found, is a
good starting point for most cell types.
______________________________________________________________________________
Date: Tue, 02 Sep 1997 12:07:00 -0400
From: ROBERT ZUCKER <ZUCKER.ROBERT@EPAMAIL.EPA.GOV>
Please check our publication in Exp Cell Research
Elstein and Zucker 211: 322-331 1994 Comparison of Cellular and
nuclear flow cytometric techniques for discriminating apoptotic
subpopulations.
We compare HO 33358 and HO 33342 on thymus cells and various
factors influencing the uptake of these dyes.
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