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> I'm doing research on polyploidy in several plant species and
>have been using flow to determine ploidy level. In order to quantify and
>compare, I have been running CRBC's as an external standard. After every
>five samples run, I run the CRBC's and adjust the voltage to get the CRBC
>....<snip>
> Some of our samples have been giving us consistently weird
>results, and if a non-linear shift is possible, that would explain what
>....<snip>
It is EXTREMELY difficult to get your system stable enough to get accurate
comparisons with an external standard; You have to make sure that everything
is exactly the same from tube-to-tube and from test-to-standard.
A difference in dye:DNA ratio will show up as a peak shift, and in my opinion
you can only measure that shift with an internal standard.
If you are needing to change the voltage to correct peak shifts, then either
your staining is irregular or your machine is unstable!
If either of these is true, then you can't assume a peak shift from sample to
sample truly indicates a difference in DNA content.
The answers:
a) find some suitable cells to use as an internal standard.
b) the internal standard cells should be prepared and stained WITH the test.
c) carefully control the ratio of cells:dye.
(assuming a constant DNA content per cell.....)
Good Luck!
Joseph.
Joseph Webster (O.I.C. Flow Cytometry & Communications) ===
Centenary Institute of cancer Medicine & Cell Biology / \
Locked Bag No.6 || o o ||
Newtown, NSW 2042 || | ||
AUSTRALIA. | \___/ |
Ph: 61-2-565-6110 \ /
Fax: 61-2-565-6101 |||||
E-Mail: J.Webster@centenary.usyd.edu.au
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