Wolfgang.Schechinger at med.uni-tuebingen.de
Thu Feb 3 04:29:41 EST 2000
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
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,
> 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
*unsolicited mail is *NOT* appreciated, usual disclaimers apply
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