[Cytometry] Aria-II 100um. nozzle stability?

Guzik, Lynda GuziLJ at ccm.upmc.edu
Thu Feb 2 09:10:49 EST 2012


Steffan,

You're brilliant!  Thank you for the explanation.  I use the 100u tip almost exclusively and have few problems.  Most of the problems I see these days are when I move from a small nozzle/high pressure to a larger nozzle/low pressure.  I don't see the problems when I move from a large nozzle/low pressure to a small nozzle/high pressure.  I always figured it had something to do with stabilizing the pressure in the tank, but your explanation makes perfect sense.  Now I know how to deal with this in the future.  Thank you!

Lynda
412-648-8660
guzilj at upmc.edu


-----Original Message-----
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Schmitt, Steffen
Sent: Thursday, February 02, 2012 7:00 AM
To: Cytometry E-Mail-Liste
Subject: Re: [Cytometry] Aria-II 100um. nozzle stability?

Hi Zip, Richard and alltogether,

We also had issues sorting with 100 um nozzles on all of our 3 Arias (Iiu, II, and III) a while ago, because we (have to) change nozzles and pressures frequently during a day (2-3 times if necessary).
But besides Vodoo, we find something else to be important and since then we nearly observe no problems anymore.

Our problems already began, that we startup the instruments early in the morning and then waited a while, till we set them up for sorting.The fluidic startup procedure is done with higher pressure than the 20 psi  we use for 100 um nozzle. So there was enough time to solve gas in the PBS tank and this was degasing after setting up the pressure to 20 psi for the sort (we could even see the bubbles sometimes in the tubings next to the cuvette).

Now we refill our tanks over night and label them with a 20 psi-stripe. As soon as we have an 100 um sort, we take a new tank and immediately setup the 100 um 20 psi sort layout.
If the same tank got to 85 um or 70 um, we take off the 20 psi stripe and know, that this tank has to be degased before it can be used for 100 um nozzle again.

With that in mind, we solved the instability and transferred vodoo into an easy organizatorical scheme.

This also explains to me, why operators have little to no problems with 100 um sorts, when they do it regularly or exclusively. They simply don´t give the system enough time to solve enough gas, which can be degas at lower pressures.


Best for you all

Steffen


Am 01.02.2012 18:02 Uhr schrieb "Richard Hastings" unter <rhastings at kumc.edu>:

Hi All,

We, too, had stability problems with the 100 um and 130 um nozzles on our Aria IIu. BD gave us a new 130 nozzle and after a few service calls, we were able to get dependable and consistent drop profiles and more importantly, the instrument sorted reliably. Our problem was the pressure regulator. Once our engineer replaced it, the instrument sorted like a dream. We have instituted quality control procedures that seem to help our larger nozzle sorts:
1. We 'burp' our sheath tank every night to prevent bubble formation in the sheath, and at lower pressure, make sure the lid is tight on the tank.
2. We sonicate our nozzles for 10-15 minutes in DI before we install them in the instrument.
3. We clean the bottom of the flow cell with Texwipe swabs dipped in DI.
4. We clean our flow cell with 50% Contrad in DI, followed by DI only.
5. Once we put a large nozzle on the instrument we let the stream run for 20-30 minutes to allow the stream to equilibrate.
6. We sort at a plate voltage of 1600 for the 130 um nozzle and 3800 for the 100.
7. We have a unique CST configuration for every nozzle size, pressure and neutral density filter combination we sort with.
8. We keep an Excel spreadsheet that we tabulate all our settings for each sort, document any problems, repairs or issues and insert the PDF report of the data. We have found this to be an excellent starting point for troubleshooting the instrument because we know how it has been performing over time. Our default 100 settings are Frequency ~17, Amplitude ~36, First drop 153, Target Gap 10 at a pressure of 20 PSI.
9. If we suspect that our large nozzle sort set up is unreliable, we do a test sort with 20 um Polyscience beads (Fluoresbrite Yellow Green Microspheres cat # 19096-2 mixed with unlabeled Polybead Microspheres cat # 18329-5). The Yellow Green beads are hydrophobic and will stick together at the bottom of the tube, when we sort the beads we dilute them in 0.1% Triton in PBS. If the instrument sorts the beads precisely, we next try to determine if our customers' sample prep or the sample itself is the problem. (Always a touchy matter!)

I learned to sort on a FacsVantage, so when I first started sorting with an Aria, it was the quite the culture shock. I miss many aspects of the Vantage; the oscilloscope, the hands on optical bench, the x, y, z controls, and I think the Vantage is easier on cells than the Aria is. I do not miss setting laser delays, or playing with the switches on the boards or spending half a day troubleshooting a problem. I have learned to appreciate the Aria, I just wish it wasn't such a closed box. I like to tinker.

Thanks, Rich




Richard C. Hastings
KUMC Flow Cytometry Core Lab
3901 Rainbow Boulevard
Kansas City, KS 66160
913-588-0627
rhastings at kumc.edu
http://www.kumc.edu/flow/


>>> "Campos-Rivera, Juanita GZ/US" <juanita.campos at genzyme.com> 2/1/2012 8:11 AM >>>
Zip,

You are not alone. One of our upgraded AriaIIs had been tremendously
difficult getting the 100-nozzle stability. After 8 different nozzles
and 3 new flow cells we are able to sort using the 100. The problem I
believe resided on the pairing between the nozzle and the flow cell, so
this last flow cell is functioning and I am able to sort. This
difficulty occurred on one of two AriaIIs.

Juanita



Juanita Campos Rivera

Flow Cytometry Core Facility

Genzyme a Sanofi Company

49 New York Avenue

Framingham. MA 01701

(508)270-2523 office 5520

(508)270-4565/4526 lab room 5225 49NYA



Our mission is to discover and deliver transformative therapies for
patients with rare and special unmet medical needs, providing hope where
there was none before.



-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Kruger Gray,
Huw
Sent: Tuesday, January 31, 2012 4:42 PM
To: 'cytometry at flowcyt.cyto.purdue.edu'
Subject: [Cytometry] Aria-II 100um. nozzle stability?





[cid:image002.gif at 01CCE036.9DABA770]



Greetings from sunny southern Florida,



     We have been experiencing difficulty with stream stability using
the 100um. nozzles on both our Aria-II and Aria-IIu, despite much
juggling of sheath pressure, etc. However, our trusty old Aria-I sorts
using the larger nozzle with little difficulty. Even our tame local
service engineers have had similar experiences with us. So, are we
alone, or might any of you have some potentially useful suggestions for
sorting on an Aria-II with these larger size nozzles? We have not even
considered trying the 130um. nozzle...!!!



Many thanks,



Zip.



[cid:image005.jpg at 01CCE037.3B2E1280]



Huw S. ("Zip") Kruger Gray, Ph.D.

>>>--->



Director, Flow Cytometry Core Facility,

Sylvester Comprehensive Cancer Centre,

Miller School of Medicine,

University of Miami,

RMSB. 7167 (office); 3061 (laboratory),

1600, NW. 10th. Ave.

Miami, FL, 33136.

305-243-5019 (office),

305-243-5571 (lab),

305-213-3325 (mobile).

www.sylvester.org/flowcytometry<http://www.sylvester.org/flowcytometry><http://www.sylvester.org/flowcytometry>



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