[Cytometry] COPAS (or similar) large particle sorter
rpulak at unionbio.com
Mon Apr 29 17:21:32 EDT 2013
One other point to ponder- some adipocytes, and depending on where the sample is coming from it can be "many adipocytes", are greater than 100 microns across, some more than 200 microns across. So working with the conventional, smaller diameter flow cell channels results in data that is selecting against the true size distribution of adipocytes. Suggests certain caveats to the data collected. -Rock
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Cris Bare
Sent: Sunday, April 28, 2013 10:07 AM
To: cytometry at lists.purdue.edu
Subject: Re: [Cytometry] COPAS (or similar) large particle sorter
By "modules" could the inference possibly be the old Macrosort option for the Vantage? If I remember they had 300 and 400 micron nozzles in that, but I've never seen anything larger than the 200 put to use.
As to the concept of running an Aria without any nozzle in place as Dave alludes, I've tried this on a couple systems just to see if I could get it to work easily. But I never optimized or ran an actual sort.
I do remember some discussion about it a few years ago and the key was it's a blind sort. You can't see the break off, thus you can't use DiVa Sweetspot. You need to manually set delay by panning values onto a slide and finding the most beads per puddle. Then run the sort and watch the deflection for fanning as an indicator of moving breakoff.
On Apr 27, 2013, at 9:11 AM, Mikhail Roshal <mir9079 at med.cornell.edu> wrote:
> Hi David,
> Did you give your 130 a shot with the adopocytes. I occasionally sort HRS cells which are about the same size and get half way decent yield and viability. Obviously a bigger nozzle would be very attractive for me as well.
> On Apr 27, 2013, at 7:18 AM, "David Haviland" <davidspurdueemail at gmail.com> wrote:
>> I'd be interested in that... I have a client who has asked about sorting intact adipocytes that can range, so I'm told, from 30-50 microns making the 130um for my Aria out of the running.
>> An anonymous tip suggested I might be able to get a break off with an Aria without a nozzle in place. So in following his suggestion(s), without a nozzle, I slowly brought the Aria down to 6.1 psi and still had a stream. At that point I was able to get curves in the stream of "want to be droplets" but there were no conditions running up and down both Amp and Freq ranges by which I could get a droplet break off in the window.
>> Any attempts to drop the pressure below 6.1 psi resulted in the stream collapsing and an ensuing mess. I gave up the ghost after about 2 hours of futzing with it. I may revisit it if I end up with a free afternoon. I have inquired with BD about a 150 or 200 um nozzle but requests have gone unanswered.
>> David Haviland, PhD
>> MHRI Houston
>> Sent from my iPad
>> On Apr 19, 2013, at 8:18, Rachael Walker <Rachael.Walker at babraham.ac.uk> wrote:
>>> By coincidence to this thread, I was told this morning by a scientist I was showing around the lab, that there are modules available to add onto existing sorters to sort large particles such as c.elegans. Does anyone know if there are such modules available?
>>> I thought on a conventional sorter you can increase a nozzle size up to 200um and if want to sort anything larger you need a Copas.
>>> Sent from my iPad
>>> On 19 Apr 2013, at 14:04, "Pulak, Rock" <rpulak at unionbio.com> wrote:
>>>> Hi Rosemary and Vanta,
>>>> The Aria is OK for eggs/embryos and the first larval stage. Anything bigger (second, third, fourth and Adults stages) starts to become too big for the 100 micron nozzle and the generation of droplets for sorting. Also, there are a lot of other particles, (for example, the cuticles of molts) that can clog the small 100 micron flow cell. Despite these limitations, it does work well for that one early slice of the animal's life cycle. The larger flow cells of the COPAS instruments and the method of sorting, overcome these shortcomings.
>>>> Best regards,
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