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I am in the process of setting up our Elite for chromosome analysis. We have
rebuild our flow cytometer in such a way that it is fitted with four lasers
now. On the optical bench are two water-cooled argon lasers (Spectra
Physics) for UV and 457nm excitation, a 25 mW air-cooled argon laser also
from Spectra Physics and the standard He-Ne laser from the Elite. The UV
laser is closest to the flow cell, second is the 457, third the air-cooled
488 and fourth the 633nm beam. We use Hoechst/Chromomycin A3 staining.
Although, the technique seems to work now in principle, there are still a
few questions. If anyone could help me, it would be most appreciated.
In our system the UV excited fluorescence signal (Hoechst) is the upper beam
for the gated amp and at the moment we collect emission at PMT2 or PMT3
using a 470nm LP. Which filters are standard for Ho/CA3 staining and what
laser power should be used for UV and 457 excitation? What about flow cell
and beam shaping optics; has anyone experience with chromosomes and an
Elite? I have aligned the system with DNA check from Coulter, but maybe it
is better to use UV beads?
Is there an established setting up protocol for this assay? If anyone has
any ideas, I would be pleased to hear them.
Thanks
Piet
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Piet EJ van Erp
University Hospital Nijmegen
dept. of Dermatology
P.O. Box 9101
6500 HB NIJMEGEN / The Netherlands
E-mail: P.vanErp@derma.azn.nl
Voice: +80-613548
Fax: +80-541184
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