Ethanol fixsation and sub-G1 population

Ulrik Stervbo ulriks at ruc.dk
Thu Jun 1 03:11:27 EDT 2006


I just realised, that in this laboratory, only 99% ethanol is
availible. Will this be a problem?

I have notices that past-postings emphasised avoiding using 99% ethanol.

Thanks
Ulrik

On 5/31/06, McCloskey, Thomas <thomasm at nshs.edu> wrote:
>
>
>
> Hi Ulrik,
>
>	  I would recommend the following:
>
> 1]  run your subdiploid apoptosis assay on a linear scale
>
> 2]  omit the formaldehyde and use only ethanol for fixation
>
> You will get tighter peaks and better discrimination of apoptotic cells with
> ethanol only.  And yoou will be less likely to include noncellular PI
> stained events if you use a linear scale.  The goal of this assay is to
> quantify cells with reduced DNA content, without including other events that
> have low PI fluorescence.  So yuou have to be careful with discriminator and
> gate setting s to achieve accurate results.  I would alos recommend looking
> at PI stained cells under a fluroescent microscope and making sure the %
> apoptotic match the flow data.
>
> Good luck,
> Tom
>
>
> *****************************************************************************
> Thomas W. Mc Closkey, Ph. D.
> Scientist in Charge, Flow Cytometry
> The Feinstein Institute for Medical Research, North Shore University
> Hospital
> Assistant Professor of Pediatrics, New York University School of Medicine
> Biomedical Research Center, 350 Community Drive
> Manhasset, Long Island, New York 11030
> ph:  516-562-4844 [office], 516-562-1084 [lab]
> *****************************************************************************
>
>
>
>
> >>>-----Original Message-----
> >>>From: ulrik.stervbo at gmail.com
> >>>[mailto:ulrik.stervbo at gmail.com] On Behalf Of Ulrik Stervbo
> >>>Sent: Wednesday, May 31, 2006 3:36 AM
> >>>To: Cytometry Mailing List
> >>>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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-- 
Ulrik Stervbo
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Germany

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