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Subject: FACSAria nozzle size ANSWERS
Date sent: Thu, 3 Feb 2005 08:31:27 -0600
From: "Stingley, Robin L" <StingleyRobinL@uams.edu>
To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
As requested, I’ve listed the answers I received to my question (below). Thanks to everyone who answered! The sort went well, and I’ve had better luck stabilizing the 100um nozzle/low pressure!
Hello Flowers,
I’m relatively new to flow, and am just starting to sort for customers with the Aria.
A new customer wants to sort mesenchymal stem cells into 96-well plates. He doesn’t know the size of his cells, but says they are similar to fibroblasts, and that once trypsinized, they aren’t very big. I don’t really want to have to change the nozzle in the middle of his sort because I have so much trouble getting the stream to stabilize with the 100 micron nozzle. Does anyone have any idea which nozzle will be appropriate for these cells?
Also, a table in the manual implies that sorting with the ACDU requires the 100 micron nozzle and low pressure. In class we sorted beads with the 70 micron nozzle on high, I think. Was this just because we were sorting beads rather than cells, or can the high pressure be used to sort cells into 96-well plates?
Thanks,
Robin
Robin Stingley, Ph.D.
Flow Cytometry Core Facility
Department of Microbiology and Immunology
University of Arkansas for Medical Sciences
Slot 511, Rm B504
4301 West Markham Street
Little Rock, Arkansas 72205
Phone: 501-686-6927
E-mail: StingleyRobinL@uams.edu
http://www.uams.edu/flowcytometry/
Answer 1:
Hi Robin,
I’m an Aria newbie myself, and like you I used to have a lot of trouble stabilizing the stream with the 100 micron nozzle. I learned to always do two things:
1. Start up the stream on High pressure and let it run for at least a minute, then switch to Low pressure.
2. Adjust the Frequency within the range 25-32 kHz to stabilize your stream.
Mine runs best at 28 kHz instead of the default 30 kHz.
Hope these help!
Richard
_
Richard Josephson, Ph.D.
National Stem Cell Resource American
Type Culture Collection
703-365-2700 x2321 10801 University
Blvd.
Manassas, VA 20110
Answer 2:
I sort regularly with the 100um nozzle and the ACDU. My base setup is 20psi/40khz. I’ve gotten the 100um tip to work at up to 50psi with the ACDU, but have not gone higher (no need).
Dave
Answer 3:
Robin,
Regarding nozzle size . . . a rule—a good ratio of cell size / nozzle orifice is about 1 / 7. So, you’ll see good performance with the 70u nozzle when analyzing cells up to about 10-12u in diameter. After that, there will be an advantage in going with the 100u nozzle. How to know? . .
. if your test-generated and AccuDrop side streams look good, but when you start running your cells the side stream quality degrades, you probably need the larger nozzle.
Regarding your sort question . . . you can sort to plates with either nozzle.
Recovery of cells (number of cells per well) can be better at lower pressures.
I recommend either Medium settings with the 70u nozzle, or Low with the 100u nozzle.
If you have a good stable stream with the 100u nozzle, that’s good . . . sort to plates with it.
I’ve also seen an improvement to cell-to-well efficiency by setting pressures at the high end of the range. So, for Medium, try 40-45 psi—for the Low setting, 25 psi seems to work better.
MAK.
Mark A. KuKuruga
BD Technical Applications Specialist
BD Biosciences
tel: 619-820-0751 fax: 443-836-9165
E-mail: mkukuruga@bd.com Website: http://www.bd.com
Answer 4:
I would “size” the sample by comparing a drop under a microscope against beads of known size(s). My guess would be the 100 um would be required, and that the 70 is too small for best drop characteristics. Particle size impacts droplet breakoff, so the actual size of the cells will determine best nozzle size.
Joe
Joseph Trotter
Director, Cytometry / Research and Development
Advanced Technology Group
BD Biosciences - Pharmingen R&D tel: (858) 812-8946 cell: (858) 449-7338 fax: (858)
812-8979
10975 Torreyana Road, San Diego, CA 92121-1106
USA
E-mail: Joe_Trotter@bd.com Website: http://www.bd.com
Answer 5:
Hello in Arkansas.
You need the 100 for sorting these cells.
We run the 100 most of the time. The only time I switch to the 70 is when I have a bunch of smaller cells.(> 10^8)
Jim Houston
St. Jude
Memphis
Answer 6:
HI Robin
I have no experience with fibroblasts or mesenchymal stem cells, but we routinely sort out CHO cells which is about 10-15 µ. We sort at medium pressure with a 70 µ nozzle. People more experienced than I would probably use a 100 µ nozzle for a start, but I see up to 70% surviving cells in a 96 well plate after single cell sort (and this is good!!!). ACDU does NOT require 100 µ nozzle. What it requires is good stable side streams (stable breakoff point). The material you are sorting means a lot to this end; I would not hesitate to sort PBMC into 384 well plate, but CHO cells are terrible, and I definately would not use more than 96 well plate.
If your 100 µ nozzle is difficult to stabilise, you can do several things: Run a couple of long clean ethanol to drive out residual air (it works!). use only adjustable nozzles, and adjust untill it behaves (e.g Ľ turn a time for a start). Finally, BD had to exchange my flow cell twice,and I can assure you that there is a huge difference. Then you should not have any troubles with 100 µ. Of course it is more fragile than on medium or high pressure, so no bumping around the machine while it is running.
hope this is helpful
· J
Jesper Kastrup, Ph.D.
Scientist
Symphogen A/S
Elektrovej, Building 375
DK-2800 Lyngby
Denmark
+45 4526 5076 (direct)
+45 4526 5050 (company)
+45 4526 5060 (fax)
Answer 7:
Hello Robin,
I routinely sort cells in 96 wells plates with the 70um nozzle and high pressure. It’s not a problem. Depending on the cells behavior towards this pressure and the nozzle to cell size ratio (big cells do not particularly like small nozzle), you might want to make a few tests to be sure that these particular cells work fine with such a settings.
\Fred
Answer 8:
Robin,
I sort mesenchymal stem cells on my Aria and the 70um nozzle is not appropriate for my MSCs. I have to use the 100um nozzle due the size of MSCs. If i use the 70 I notice in my post sort analysis that I have a lot of dead cells and debris, most likely pieces of cells that resulted from too small a nozzle with large cells.
Regarding the ACDU. I would do ACDU sorting at low pressure with the 100um nozzle. I found that everything that I learned in training is good for the overall basics of the instrument. However, in training we used beads and the beads are uniform and the Aria is perfect at sorting beads; cells are not as ideal when sorting. It took me a couple of weeks to asses which conditions are optimal for each cell type. If you have any more questions please email me and Ill try to help you out.
The Aria is usually a fantastic machine or an incredible headache, no grey area.
Adam Palazzo
Perlingeiro Lab
Center for Developmental Biology
University of Texas Southwestern Medical Center at
Dallas
214.648.7354
Answer 9:
Hi,
These cells will require a 100um nozzle or even a 130um but I believe the Aria doesn’t go that high. You can’t use high pressure with a 100um nozzle because at high pressure with that diameter you wouldn’t be able to produce drops.
With a large stream diameter pressure fluctuations are more pronounced and consequently you need to be positive all air bubbles are removed from the fluidics to get a stable drop delay.
On the Vantage/SE you can perform single cell sorting into well plates with any size nozzle but you must be in an appropriate sort mode so that only single cells are diverted from the stream i.e. one drop packet sort condition. Again I don’t know about the Aria but in DIVA software this means a 0 yield and 32 purity mask and possibly a phase mask to force feed the desired cell into the center of the drop.
Gene Pizzo
Answer 10:
Hi Robin-
I would sort with a 100 um nozzle on low pressure. If the cells are similar size to fibroblasts and are fragile you wouldn’t want to attempt anything else smaller. Hope that helps.
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Hope Albertz
Flow Cytometry Core Lab - Blood Center of
Wisconsin
414-937-3843/3849
