[Cytometry] Influx, Astrios, Reflection? WAS Re: Aria II or an Influx

Elliott, James james.elliott at csc.mrc.ac.uk
Tue Feb 16 05:16:24 EST 2010


Hi Jan

Actually we've just been chatting with Joe Trotter on this by email. Most likely is the latest Spigot 6.1.9 software from the Cytopeia website will help as this fixes some purity bit mask bugs. We have always regarded Spigot as by far the most likely culprit, yet I think it is a failing that BD support at this level is patchy and there is no coherent strategy for making us aware of these kinds of problems and fixes. For example, we have pressed them in the past about software updates but have been told that these probably won't help and will most likely cause more problems than they're worth. That Amstel has been around the corner for a long time a disincentive to push our doubts about Spigot too hard. It would help if engineers were better trained in cytometry/software as well as the hardware.

We cross-check purities of collected samples between all machines, so no, it is not that contaminants from the Influx are small particles missed by the others. That would be too easy!

Best

James Elliott
Phil Hexley

MRC CSC 
London

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of BAIJER Jan 212040
Sent: 16 February 2010 09:33
To: Cytometry List
Subject: Re: [Cytometry] Influx, Astrios, Reflection? WAS Re: Aria II or an Influx

Hello all,

I really tried, but I cannot not react. Sorry ... I'd like to respond to
both the cited message, as well as the earlier message in the same thread by
David Ko.

James, I would be more than interested to find out the actual technique
you're using to determine your post-sort purity to be between 99.5% and 100%
on an Aria, and somewhat lower (!!!) on your InFlux. Excuse me all over the
place, but this smells. What, in your opinion, could possibly explain such a
difference ?! I think that both sorters would give 100% purity, depending on
sort modes, but most of all, depending on the biology that is done up front.
Since predominantly that biology is limiting, because of aggregates
produced, cell death/fragmentation induced, crappy labeling strategies used,
etc. etc. etc. etc., day to day sort purities are not 100%. Is  your InFlux
equipped with the small particle scatter detector ?! Part of the answer
might be that you're actually seeing small stuff on the InFlux that you
would never see elsewhere. On the InFlux, we actually sort at zero threshold
level (we can't do this on the MoFlo). Bottomline: I do not believe that
these two sorters would give significant differences in sort purities
obtained.

David, related to the above, you are really challenging me (and I would hope
many others), by saying that the Aria stands out for its high purity at high
sort speeds. First of all, 15,000 per second is not fast (although it is
probably as fast as an Aria will ever go). Aria is totally incapable of
pushing extremely highly concentrated samples, and it will never obtain the
flow rates needed for real high speed sorting. We run samples in excess of
2x10e8 cells per ml to obtain significantly higher sort speeds. The only
machines capable today of doing this are InFluxs and MoFlos (haven't seen
Astrioss). We're sorting murine stem cells at flow rates of approaching
50,000 (we reject quite some, I know ...) for several years now, and are
really impressed by the quality of the work provided by our recent InFlux.
Our five-laser machine is easily aligned, and calibrated by our "equally
new" technician. Like I said, we're impressed ...

You'd better consider yourself lucky that I didn't start talking about the
InFlux sound optical design providing excellent sensitivities ...

Jan

--
Jan Bayer, Ph.D.
Flow Cytometry Platform
CEA-FAR-DSV-iRCM
Route du Panorama BP 6
92265 Fontenay-aux-Roses Cedex

France
TEL: +33 (0)1 46 54 97 51
FAX: +33 (0)1 46 54 91 80
--


> From: "Elliott, James" <james.elliott at csc.mrc.ac.uk>
> Date: Mon, 15 Feb 2010 13:10:06 +0000
> To: Cytometry List <cytometry at lists.purdue.edu>
> Subject: Re: [Cytometry] Influx, Astrios, Reflection? WAS Re:  Aria II or an
> Influx
> 
> Hi all,
> 
> We have an Aria II, Influx and a Vantage/DiVa. As others have said, the
> obvious advantages of the Influx are the flexibility in laser configuration,
> installation and being able to move around PMTs, changing sample lines etc.
> One of the principal disadvantages, at least with our Influx in our hands is
> that we can't get purity as high as with either DiVa or Aria. Thus, a typical
> sort for us where we'd expect 99.5-100% purity with the Aria, we'll get
> 99-99.5% on the DiVa but only just over 98% on the Influx (grateful for any
> suggestions from more successful Influx users!). As mentioned elsewhere, 4 way
> sorting is poor. Spigot software (soon to be replaced for ages!) is also far
> more cumbersome than FACSDiva.
> 
> When time comes to replace the DiVa we'd consider an Astrios, yet information
> is still scant. How reliable is 6 way sorting, for example?
> 
> Best
> 
> James Elliott
> Phil Hexley
> 
> MRC CSC
> London
> 
> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu
> [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Adrian Smith
> Sent: 14 February 2010 08:48
> To: Cytometry List
> Subject: [Cytometry] Influx, Astrios, Reflection? WAS Re: Aria II or an Influx
> 
> 
> On 13/02/2010, at 7:52 AM, Moody, Pamela wrote:
> 
>> Hi Everyone,
>> We currently have an Aria II SORP and a FACSVantage DiVa in our
>> facility.  We are trying to decide between an additional Aria II SORP
>> with 5 lasers or an Influx.  If anyone has experience with both of
>> these
>> instruments I would appreciate hearing some feedback on them both.
>> Specifically why you would choose one over the other.
>> Thanks for your help.
>> Regards,
>> Pam
>> 
> 
> 
> Hi Pam,
> 
> Why limit yourself to the Influx?
> 
> We're looking at a new sorter with more features and capabilities than
> our Aria II and the BC MoFlo Astrios and iCyt Reflection are on our
> list alongside the Influx.
> 
> Key things we are after are increased speed/throughput, more laser
> lines, biosafety containment etc.
> 
> Should be an interesting decision :)
> 
> (and if anyone has experience or comments they would like to share (on-
> list or off) about iCyt Reflection or MoFlo Astrios (or XDP) I would
> love to hear them).
> 
> Regards,
> 
> Adrian Smith
> Centenary Institute, Sydney, Australia
> 
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