Cell Cycle Analysis on FACSAria

Derek Davies derek.davies at cancer.org.uk
Sun Feb 10 16:29:58 EST 2008


Hi Jodene,

The Aria does have a smaller spot size than many other cytometers (its about
9 microns I think but am prepared to be corrected!) but you certainly can
run DNA analysis on it. Indeed we have also sorted SP cells which demands a
good precision of the G1 peak. There are some issues using width which is a
calculated parameter in DiVa but that only really affects very large clumps
or reduplicating cells. If you are interested in DNA alone you could trigger
on the DNA signal, in addition to running at a lower pressure.

I would agree with Willem that preparation is a key factor and with Ian that
sometimes the Aria does require a bit of a tweak of the alignment (which is
why I use the LSRII unless I need to sort them!). If I am running DNA
samples, these are the best things to check alignment with, not beads.
Having a stock of ethanol fixed cells such as Jurkats is a good idea!

Derek


On 8/2/08 9:49 am, "Ian Titley" <Ian.Titley at icr.ac.uk> wrote:

> 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
> 
> Always plan ahead. It wasn't raining when Noah built the ark.
> 
> Go to http://calendar.yahoo.com/facslabfels to check the Flow Cytometry
> Facility online
> appointment calendar.
> Jodi Moore, PhD
> Flow Cytometry Facility Manager
> Temple University, School of Medicine
> Fels Institute of Cancer Research and Molecular Biology
> Room 315, AHP
> 3307 North Broad Street
> Philadelphia, PA 19140
> 
> Core Lab: 215-707-4119, Room 220
> Office: 215-707-7709, Room 315
> Sorter: 215-707-4043, Room 221A
> 
> Temple Flow Core Facility Website:
> http://www.temple.edu/ovpr/flow_cyto/
> 
> The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company
> Limited by
> Guarantee, Registered in England under Company No. 534147 with its Registered
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-- 
***************************************************************
Derek Davies, FACS Laboratory, London Research Institute,
Cancer Research UK, 44 Lincolns Inn Fields, London, UK.

Tel: (44) 20 7269 3394
FAX: (44) 20 7269 3479
mobile: 07790 604112
e_mail: derek.davies at cancer.org.uk
Web Page: http://science.cancerresearchuk.org/sci/facs/

In tenebris lux    
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