Re Re DNA analysis from cardiomyocytes

Dennis_Young@CIS.ucsd.edu Dennis_Young at CIS.ucsd.edu
Thu Apr 20 13:05:00 EST 1995


Andreas
Now that you have provided more details, I think we still need to know more.
You didn't mention the use of RNAse. It's been a few years since I've looked at 
adult rat cardiomyocytes, but I don't remember staining for DNA. With PI (or EB)
I would ALWAYS use RNAse (boiled, renatured at 100 ng/ml final for 30 min at 37 
C ).
 With UV, I use DAPI (I think 2 microg/ml + 0.2% Triton-X in PBS- UNFIXED!) and 
never used RNAse. It gives better CV's than the Ho33XXXs.
It sounds like a problem with accessability of the DNA dyes.
How are you staining for RNA and the proteins? Do you include DNAse control?
Sounds like the nuclear membrane of cardiomyocytes is not as porous as other 
cells.
What about just DNA/RNA (Acridine Orange)?


*****************************************************************************
* Dennis J. Young                            Voice : (619) 543-3928         *
* Flow Cytometry Core Facility               FAX   : (619) 543-7487         *
* University of California, San Diego  USA   e-mail: djyoung at ucsd.edu       *
*****************************************************************************

I think I have to explain my problems more exactly.

I use a Coulter Epics Elite ESP with a 325 nm HeCd, a 488 nm Argon and a
633 nm HeNe laser. We have also a gated amp for two laser excitation. As
we have good experience measuring DNA of other cells as fibroblasts, we
tried the same protocols for the cardiomyocytes. We tried ethanol (70-90%,
4 to -20 degree Celsius, 1 hour up to 24 hours) and formaldehyd /
paraformaldehyd (1-4%, 15min - 24 hours) as fixation. In the case of
formaldehyde, we used Triton X-100 or NP-40 for lysing/opening the cells
afterwards. We used the Hoechst 33258/33342 dyes and Ethidium Bromide as
DNA stains. But with all trials, we got bad results in regard to the
DNA measurements. When we use the fluorescence microscope, we see no
uniform stain of the nuclei of our cardiomyocytes. So the problem is a
staining problem.
When I stain a freshly preparation of cardiomyocytes, we see also other
contaminating cell populations by foreward and sidescatter parameters.
Gating on these cells - fibroblasts, endothelial cells or lymphocytes -
gave very nice DNA histograms. So, it is a problem of these specific
cells. Freshly prepared cardiomyocytes are huge cells - 10 fold bigger
than fibroblasts by cell volume  and 100 -150 microns long -, have
2 nuclei and a mass of RNA and protein.
We want to measure DNA and RNA / protein simultaneously, but at the moment,
failed to analyse the DNA.
I would appreciate any comment, advice or idea.

Andreas


Andreas Simm
Physiologische Chemie II
Biozentrum, am Hubland
D-97074 Wuerzburg
Tel.: +49 - 931 - 888 4128
FAX.: +49 - 931 - 888 4113



More information about the Cytometry mailing list