What's the shelf life of PFA-fixed cells?

Prussin, Calman (NIH/NIAID) [E] CPRUSSIN at niaid.nih.gov
Wed Feb 8 13:46:24 EST 2006


Vladislav,

 I wish I had an intelligent response to your very good points.

> I just wonder if fixed cells require intracellular cryoprotectants like DMSO?
>> I have not tested this and am not aware if anyone else has.

> Another question is whether 10% DMSO is a tested optimum or a replicate of 10% DMSO for
freezing live cells? 
>> Just a replicate of the 10% DMSO used to treat live cells.

Perhaps someone else on the list has actual data to make me look less clueless? 

Calman
> ----------
> From: 	rozenkov at netscape.net
> Sent: 	Wednesday, February 8, 2006 6:47 AM
> To:	Prussin, Calman (NIH/NIAID) [E]; cytometry at flowcyt.cyto.purdue.edu
> Subject:	Re: What's the shelf life of PFA-fixed cells?
> 
> Dear Calman,
>  
> It is a really good method to store cells if they fully preserve their phenotype.
>  
> I just wonder if fixed cells require intracellular cryoprotectants like DMSO?
>  
> Another question is whether 10% DMSO is a tested optimum or a replicate of 10% DMSO for
freezing live cells? 
>  
> I think freezing/thawing techniques of live cells using DMSO are based on osmotic
gradient rules, so in the beginning of gradual freezing extracellular DMSO is at 10%, but
by the eutectic point (which is about -64 grad C, i.e. below -80C) DMSO replaces
cytoplasmic water and reaches some 70% intracellularly when vitrification occurs. I guess
this does not happen in fixed cells, and 10% DMSO circulates freely.
>  
> Thanks.
>  
> Vladislav Rozenkov, M.D., Ph.D.
>  
> -----Original Message-----
> From: Prussin, Calman (NIH/NIAID) [E] <CPRUSSIN at niaid.nih.gov>
> To: Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>
> Sent: Mon, 6 Feb 2006 14:43:16 -0500
> Subject: RE: What's the shelf life of PFA-fixed cells?
> 
> 
> We routinely store 4% PFA fixed cells in PBS/10% DMSO at -80 degrees C and they 
> are
> stable for years for immunostaining and intracellular cytokines. I have not done 
> TUNEL.
> 
> See Current Protocols in Immunology, Unit 6.24.
> 
> Calman
> > _______________________
> > Calman Prussin
> > Laboratory of Allergic Diseases
> > National Institute of Allergy and Infectious Diseases/ NIH
> > 
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> 
> > ----------
> > From:     Josef Schulze-Horsel
> > Sent:     Thursday, January 19, 2006 6:06 AM
> > To:   Cytometry Mailing List
> > Subject:  What's the shelf life of PFA-fixed cells?
> > 
> > Hello FLOWers,
> > 
> > I have aquestion concerning the storage of paraformaldehyde (PFA)-fixed 
> > sample, especially for later TUNEL-measurements.
> > I saw a decrease in positive cells after several weeks...
> > 
> > Do you have any experience how long such samples can be stored in 1 or 2 
> > percent PFA at 4°C?
> > Is it advantageous to wash the cells directly after fixation and store 
> > them e.g. in 70 % ethanol at -20°C?
> > 
> > What are your recommendations?
> > Thanks a lot for your advice.
> > 
> > Have a nice day!
> > 
> > 
> > Josef
> > -- 
> > *********************************************************************
> > 
> > Josef Schulze-Horsel
> > Bioprocess Engineering
> > 
> > Max-Planck-Institute
> > Dynamics of Complex Technical Systems
> > Sandtorstr. 1
> > 39106 Magdeburg
> > 
> > Germany
> > 
> > Phone: +49 (0)391 - 6110-208
> > Fax:   +49 (0)391 - 6110-571
> > E-mail: horsel at mpi-magdeburg.mpg.de
> > Homepage: www.mpi-magdeburg.mpg.de
> > 
> > *********************************************************************
> > 
> > 
> 
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