Re: Ethanol fixation and sub-G1 population

Joanne, I think your message is the best finish to the discussion on "fixSation and
sub-G1" (it has been even more annoying to see this title again and again). Really, of
the huge variety of contemporary apoptosis tests there are many assays that are simple,
easy and technically quite straightforward, while a real sub-G1 testing requires a high
level flow cytometrist, as Zbigniew and Derek explained. The problem is that a number of
people imagine themselves investigating apoptosis while in realty they are looking at
sub-cellular pieces or just can not stain properly. As you and Bill noted, high rank
journals stopped publishing sub-G1 papers several years ago, but there are still tens of
papers appearing in other journals. So, in the end of the day, it is up to each
investigator to understand what s/he is doing. Luckily, at least all members of this List
know the truth.
Regards.
Vladislav Rozenkov, M.D., Ph.D.
-----Original Message-----
From: Joanne Lannigan <jl7fj@virginia.edu>
To: cyto-inbox
Sent: Sat, 10 Jun 2006 12:19:40 -0400
Subject: Re: Ethanol fixsation and sub-G1 population


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 
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 
> 
> -----Original Message----- 
> From: Ulrik Stervbo [mailto:ulriks@ruc.dk] 
> 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 
> 
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