Brdu staining

[Wolfgang Schechinger]

Dear Dror, 

First, try to reduce the denaturation time and / or temperature (if 
you use HCl, of course ;-).

You also might want to check the BrdU cell growth assays offered from 
Roche Diagnostics (former Boehringer Mannheim). They provide a 
solution for denaturing the cells (called FixDenat) you may order 
separately from the kit. It's probably a mix of ethanol, protease and 
buffer (my own opinion). 

If you're intersted in grwoth data, you could consider measuring the 
incorporation of 3H-thymidine as an alternative. Though radioactive, 
in my eyes it is very convenient and accurate.

BTW, would you please pass over your HCl/microwave protocol? I'm just 
curious. 

When you have enough cells (let's say at least three dishes am 3 cm 
each) and you're just interested in their absolute DNA content, you 
might use dyes like CyberGreen or H33258 (aka 
bis-benzimidazol, both sold by http://www.probes.com ) which become 
fluorescent upon DNA binding. EtBr might do the job, too.

All the best, 

Wolfgang


> Hi:
> 
> I am trying to stain a monolayer of endothelial cells on a collagen
> lattice with anti-Brdu monoclonal antibody.  The assay requires a 20
> minute incubation with HCL (or microwave) to partially denature the
> DNA.  The denaturing step results in a complete destruction of the
> collagen substrate and subsequent loose of Endothelial cells.  I was
> wondering if there are methods for partial DNA denaturing which are
> not destructive to reconstituted collagen.
> 
> Thank you,
> 
> Dror Seliktar
> Georgia Institute of Technology
> 
> 
> Sent via Deja.com http://www.deja.com/
> Before you buy.

Dr. Wolfgang Schechinger, Pathobiochemistry Dept.      
University of Tuebingen, Germany
email: wolfgang.schechinger at med.uni-tuebingen.de 
wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
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