Summary Document
This document is a summary of message from the Purdue University Cytometry Laboratories Cytometry Discussion list.
http://www.cyto.purdue.edu/hmarchiv/index.htm
Considering the longevity of this topic, I believe a synopsis is warranted.
Noah
-----Original Message-----
From: Ulrik Stervbo [mailto:ulriks@ruc.dk]
Sent: Wednesday, May 31, 2006 3:36 AM
To: cyto-inbox
Subject: Ethanol fixation 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 stained
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
-------------------------------------------------
RESPONSES:
On Jun 8, 2006, at 3:47 PM, Telford, William ((NIH/NCI)) [E] wrote:
Just to add a couple of things to this good discussion...
The appearance of the sub-G0/G1 is highly variable between cell
types, and even the same cell type at differing levels of
activation. Cycling cells in particular (with less compacted
chromatin) are particularly likely to give confusing results,
especially with the accumulation of subcellular objects
(cell fragments, fragmented chromatin, other trash) at the
low threshold of instrument sensitivity. The contribution
of S + G2/M cell apoptosis to the "pot" further complicates the issue of
data interpretation. And if we can't clearly separate intact
apoptotic cells from apoptosis-associated debris, we are not really measuring
cell-by-cell apoptosis anymore.
We strongly encourage our investigators to use sub-G0/G1 ONLY as a
preliminary, qualitative indicator of cell death, not as a
quantitative assay - unless the apoptotic peak is really clear, which does not
happen most of the time. It should always be backed up with additional
assays, especially biochemical ones like caspase activation. And most
journal reviewers, grant review panels, etc. won't be impressed
anymore if DNA loss is the only criterion for measuring cell death - there are
lots of other nice flow apoptosis assays available now, some of them fairly
economical.
Enjoy,
Bill Telford
-------------------------------------------------
From: Joanne Lannigan <jl7fj@virginia.edu>
Date: Saturday, June 10, 2006 9:49 pm
Subject: Re: Ethanol fixation and sub-G1 population
To: cyto-inbox
I have to strongly agree with Bill Telford. As cytometrists we have
so many better tools for evaluating apoptosis that the use of sub-
G1 should only be used for a quick screen evaluation (if that), and in
itself is inconclusive. The problem is that there is still a
plethora of literature siting this method which many new investigators find
and believe is an appropriate assay for quantitating apoptosis.
Let's stop contributing to that list of references.
Joanne Lannigan, M.S.
Director, Flow Cytometry Core
University of Virginia
Jordan Hall Room 7065
1300 Jefferson Park Avenue
Charlottesville, VA 22908-0734
flowcytometryj@virginia.edu
(434) 924-0274 Office
(434) 982-1071 Fax
