Doublet discrimination : Aria vs Vantage DiVa
Ian.Dimmick at newcastle.ac.uk
Sat Apr 15 11:38:15 EDT 2006
I have tested the doublet discrimination on the FACScanto , LSR II ,
FACSAria, FACSCalibur using
1 Area v width
2 Area v height
3 ratio of width +height
These were compared with conventional area v width on the FACSCalibur
using BD QC particles, all gave exactly the same results, as already
stated doublet discrimination theory relies on your cells, since doublet
detection relies on a perfect spheroid model and cells are usually not
that ,therefore this theory applied to the cell, and the assumption for
the doublets to be nicely stuck one to the other in a vertical aspect
when traversing the analysis point of the laser beam is not always the
case, the further you get away from this theoretical model then the more
is the probability that you will not detect doublets.
Flow Cytometry Core Facility Manager
Institute of Human Genetics
Newcastle Upon Tyne
Ian.Dimmick at ncl.ac.uk
Tel 0044 191 2418831
Fax 0044 1912418666
(mob) 0044 7970344823
From: Yoav Altman [mailto:yoav at burnham.org]
Sent: 13 April 2006 20:20
Subject: Re: Doublet discrimination : Aria vs Vantage DiVa
I don't think there's a hard-and-fast rule about whether to use
height vs. width on the Aria vs. Digital Vantage for doublets. In my
experience, both with a Digital Vantage and a FACSCanto, the
parameters used to discriminated doublets depend more upon the
cells than the instrument.
I start out with two dotplots, FSC-A vs. FSC-H, and SSC-A vs. SSC-W.
Sometimes the H vs. A plot gives the best discrimination,
sometimes the A vs. W plot is better, sometimes either one will
do, and sometimes I use both, gating first through one, then
further gating that singlet population through the second plot.
Try it out and see what works for you. I think you'll find that cell
morphology dictates your approach more than instrument choice.
Manager, High Throughput Cell Analysis
Burnham Institute for Medical Research
10901 North Torrey Pines Road
La Jolla, CA 92037
At 8:25 AM +0200 4/13/06, Roberts Joanna wrote:
>Could anyone please offer me an explanation about doublet
>discrimination in DiVa software on the Vantage versus on the Aria?
>....inasmuch as i have believed that on the Vantage setup in Diva, one
>uses FSC-A vs FSC-H to best acheive this while on the Aria, width is
>Have I got the right end of the stick? Is it important?
>If I have got this right, could somone kindly explain to me
why this is so.
>Thank-you so much,
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