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