[Cytometry] fixation of dermal fibroblasts in suspension for storage and subsequent use in flow FISH and immunolabeling

P.Dekker@lumc.nl P.Dekker at lumc.nl
Thu Jul 23 06:02:10 EDT 2009


Dear all,

 

We are doing experiments with multiple strains of dermal fibroblasts
which are also grown under multiple conditions, hence we have to do our
experiments in batches of strains. We want to use flow cytometry to
measure cells labeled for a few proteins, some of which are cytosolic
and some of which are nuclear. Also we would like to perform flow FISH
to measure relative telomere length.

 

Since it would be convenient to be able to store fixed cells for
subsequent analysis, we were wondering what method we should use for
fixation. Up to now I have used 4% paraformaldehyde (20 minutes) for
fixation and this works well for p16-labeling, in combination with 0.2%
Triton for permabilization for 20 minutes. I think this is a rather
stringent method as many protocols use 1 or 2% paraformaldehyde for up
to only 10 minutes and then permeabilizing  with 0.05% Triton, but I
guess this also depends on whether use want to look at cytosolic or
nuclear proteins. 

 

I have also found literature stating that flow FISH can be done on cells
fixed with 1-3% paraformaldehyde and stored for up to 3 weeks
(Murell-Bussell et al., 1998). On the other hand, other people seem to
think paraformaldehyde is a poor fixative.

 

What are your experiences with flow FISH on fixed cells (or more
specifically dermal fibroblasts in suspension) and do you think there is
an optimal protocol for fixation allowing flow FISH and immunolabeling
for cytosolic and nuclear proteins (not necessarily co-labelings)?

 

Thank you in advance.

 

Regards,

 

Pim Dekker

PhD student

Leiden University Medical Center 
Section of Gerontology and Geriatrics, C2-R 
Department of General Internal Medicine 
Postbox 9600 
2300 RC Leiden 
The Netherlands 
Tel.: +31 (0)71 5261312
Fax.: +31 (0)71 5248159
E-mail: p.dekker at lumc.nl 

 



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