Cell Cycle Analysis on FACSAria

Ian Titley Ian.Titley at icr.ac.uk
Fri Feb 8 04:49:50 EST 2008


Jodi

We have a 5 laser SORP Aria that has UV, 488, 532nm and can get
acceptable DNA profiles for PI stained samples on all three lasers. It
has taken some time and considerable help from BD and their engineers to
obtain this. The blue laser which is at time position 0 has always
looked fine but UV and green +/- the extremes for time delay showed no
identifiable DNA profile when aligned by BD with their standard
alignment beads. We, collectively, found that the laser steering mirrors
for UV and green occasionally need a "tweak" to obtain good CV's but it
is usually not too far out, also that a large window extension, say 5us
or more, is needed although I realise this may have some impact if
trying to sort at high speed. I find that we also need to tweak the
steering mirrors on a known well stained DNA sample since beads do not
seem to align the machine optimally for DNA, although if aligned with
beads the BD DNA QC kit (calf thymocyte nuclei) looks fine? Current
thoughts are that large particles (ie whole cells) behave differently in
the flow cell than smaller particles beads or nuclei??? We get doublet
discrimination similar to our LSRII and VantageDiVa when the machine is
correctly set. 


Best wishes

Ian



Ian Titley PhD
Section of Haemato-oncology
Institute of Cancer Research
15 Cotswold Road
Belmont
Sutton
Surrey	SM2 5NG
UK

Tel +44 (0)20 8722 4255



>>> Jodene K Moore <drjmoore at temple.edu> 06/02/2008 14:50 >>>
Hi Flowers-I am under the impression that due to the unique beam
geometry of the Aria that cell cycle analysis is not possible
on this instrument (due mainly to the reduced spot size that
makes doublet discrimination impossible). Has anyone been able
to successfully run cell cycle on the Aria? Thanks for the
input. Any suggestions appreciated.
Sincerely,
Jodi

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