Re: Ethanol fixsation and sub-G1 population

Hi!
Thomas brought up an important issue which I'd like to ask for help on, which is gating. After looking a bit at the literature and at the archives, I have found that sub-G1 data is based on faith a lot of the time. Either faith on something someone else did ("he does not show gating, so I must believe he did it correctly"), or faith in what you are yourself doing ("MY gating is right"). Because if you change the position of your FSC-SSC and/or width vs height gate you can obtain extremely different results. Being quite laic in my scientific life as well as relatively naive regarding sub-G1 measurements, I'd love to know if there is a method to correctly gate W vs H data. 

Does any one have such a systematic method that is scientifically defendable? or is it a matter of experience (and style!)?

many thanks, 

regards,

Uriel.
Uriel Trahtemberg
MD/PhD student
The Laboratory for Cellular and Molecular Immunology
The Hebrew University - Hadassah Medical Organization
Jerusalem - ISRAEL 

"You don't need data, all you need is style!"


  ----- Original Message ----- 
  From: thomas.delohery@verizon.net 
  To: Cytometry Mailing List 
  Sent: Wednesday, June 07, 2006 6:35 AM
  Subject: Re: Ethanol fixsation and sub-G1 population


  just a brief comment regarding sub-G1 cells/material... more to emphasize rather than add anything new to the discussion.
  the diminished fluorescence of of sub-G1 (or sub-G2/M) is not necessarily or exclusively due to the loss of small DNA fragments.  
  Some of the events registering a decrease in fluorescence are cells in early stages of programmed cell death that have changes in chromatin structure leading to a decrease in fluorescence intensity prior to any loss of DNA.  For me, the most important aspect of analyzing this data is gating - not whether the data is collected in log or linear; because we all have to establish gates that provide the appropriate (fair) quantitation of cell death. One needs to eliminate the debris without eliminating the cells of interest.  Therein, lies the art versus the technique or SOP.  

  best, TomD


  Derek Davies wrote:

Anyone involved with looking for apoptotic cells by the sub-G1 method
should
print out Dr Darzynkiewicz's reply! My Lab is involved with several groups
who are interested in quantitating 'apoptosis' and one of the most important
things to get them to realise early on is that events can appear in the
'subG1' region of a DNA histogram for several reasons, DNA degradation in
apoptosis being one of them. Additionally the cells that appear in that
region in unfixed cells (after Hoechst DNA staining for example), in cells
fixed in paraformaldehyde and in cells fixed in ethanol are not necessarily
the same!

Paraformaldehyde is the way to keep the cell as it was at the point of
fixation as it is a cross-linker but isn't ideal for subG1 as firstly CVs
will be sub-optimal and secondly, cells that have fragmented - but not lost
-  DNA will show 'normal' DNA content and any subG1 events will be cells
that have physically lost DNA prior to fixation.

Ethanol fixation (we routinely use 70%) which dehydrates and coagulates
proteins will allow small fragments of DNA to be eluted post-fixation and
then these will appear in the subG1 region. However it is also important to
realise that cells entering apoptosis from late in the cell cycle may not
appear here. It is possible sometimes to see a qualitative effect on the
histogram and that is one of the reasons why a linear scale will allow you
to see this better. In these cases a subsequent or parallel experiment
looking for strand breaks using the TUNEL technique would be indicated. It
is also possible to combine this staining with surface staining, we have
done this with Alexa488 labelled antibodies which will survive the ethanol
fixation and processing.

And, as Tom said, looking at the sample under the fluorescence microscope
can be informative. Whichever cell type you are using it is also very
important to correlate results from subG1 staining with cell counts and, if
possible, proliferation assays as taken in isolation, results from a single
cytometric assay are open to misinterpretation!

Good luck!
Derek


On 31/5/06 7:42 pm, "Darzynkiewicz, Zbigniew" <Z_DARZYNKIEWICZ@NYMC.EDU>
wrote:

  While fixation in formaldehyde is required to detect apoptotic cells in
the TUNEL assay, formaldehyde should not be used in the assay based on
analysis of cellular DNA content ("sub-G1" cell population); ethanol at
50 - 80% concentration is preferred. Unlike formaldehyde, fixation in
ethanol does not crosslink DNA and thus allows the low molecular weight
fragmented DNA to be extracted from the cells when they are transferred
from ethanol to buffer or PBS, incubated with RNase and stained with PI
or DAPI, so apoptotic cells may end-up with fractional DNA content. In
fact, when cells are fixed in formaldehyde, apoptotic cells often cannot
be identified as the sub-G1 population. It is only when the apoptotic
process is very advanced and some DNA is being lost by shedding
apoptotic bodies that contain parts of fragmented nuclei, apoptotic
cells may have fractional DNA content and be distinguished as "sub-G1"
cells after formaldehyde fixation. It is also worth to notice that if
G2M cells undergo apoptosis they may end-up with a "sub-G2M" DNA content
which may locate them at the site of S-phase cells on the DNA content
histograms. 


Zbigniew Darzynkiewicz, M.D., Ph.D.
Brander Cancer Research Institute
New York Medical College
Valhalla, NY, 10595
http://www.darzynkiewicz.com/zbigniew/


-----Original Message-----
From: ulrik.stervbo@gmail.com [mailto:ulrik.stervbo@gmail.com] On Behalf
Of Ulrik Stervbo
Sent: Wednesday, May 31, 2006 3:36 AM
To: cyto-inbox
Subject: Ethanol fixsation and sub-G1 population

Hello everyone,

I would like to measure the size of the sub-G1 population on
logarithmic scale as well as distribution of cells in the cell cycle
on lineary scale, by collecting the fluroscence of PI staind cells in
FL3 and FL2.

In this laboratory, they fixate cells in formaldehyde according to a
well establish protocol for measurement of the sub-G1 population. I
understand from the archives of this list, that the broad G1 and G2
tops I see are due to the formaldehyde.

Is there any reason why one should prefer formaldehyde over ethanol
fixation? Does use of formaldehyde yield beter apoptosis data? The
focus is measurement of of the sub-G1 population, analysis of cell
cycle distributionwould just be a nice bonus.

Heres the protocol in brief:
Incubate with 2% formaldehyde on ice for 30 min
Remove formaldehyde and incubate with ethanol on ice for at least 15 min
Remove ethanol and incubate with RNAse for 30 min at 37 degree
Remove RNAse and add PI

Thank you in advance for any help/thought on this matter
Ulrik