Hello Flowers, Thanks for all the replies to my LSRII Sheath Buffer and Aria O-ring question. The original e-mail and all of the responses (minus the name of the sender) are pasted below. Here is the quick answer to each question: Do other users have surfactant in their LSRII buffer and have you seen an increase amount of cell death due to surfactant? I found out that we should be using sheath with surfactant since two of our LSRIIs have HTS systems. You are supposed to have surfactant in your sheath if you are using an HTS system. No one has seen an increase amount of cell death with surfactant. Since the cells and buffer are only together for milliseconds before entering the flow cell, I didn't think this was going to be too big a problem. Has anyone experienced an inordinate amount of aria o-ring shredding with the new amber flow cells? There are definitely other users who are also experiencing the same problem. There was a variety of suggestions in your responses but none of them helped. BD is going to come in and replace our flow cell. It's likely the reason we are shredding so many o-rings. The last time our field service engineer was in he removed the nozzle to place a scope on the machine to view the flow cell. I proceeded to shred 6 o-rings in the span of 10-minutes trying to replace the nozzle. After seeing this, our engineer has no doubt it’sour flow cell. Thanks again for all the responses, Geoff- Geoffrey Lyon Biotechnology Associate I FACS Facility Yale University School of Medicine TAC Building Rm-S613 300 Cedar Street Shipping Address: 1 Gilbert Street TAC Building Rm-S617 New Haven, CT 06519 New Haven, CT 06520-8035 (203) 785-2541 (203) 785-7949 -------------------------------------------------------------------------------- Hello Flowers, I have two different questions I was hoping you all might be able to help me with: 1) We have had quite a bit of residual air floating around our LSRIIs. I was told by a few different people, including a BD Service engineer, that a small amount of surfactant in the sheath eliminates almost all the trouble we have been experiencing with air bubbles. Has anyone else tried this? Does surfactant kill most of the cells even at low concentrations since it is a detergent? 2) Am I the only one who now sees the Aria nozzle o-rings as a single use item? It seems that ever since we got the new amber style flow cell that once we take out an o-ring that o-ring is pretty much finished. We can actual see small pieces of the o-ring shear off after removing the o-ring from the flow cell after a single time. It seems that the new flow cell is so sharp it cuts fine pieces of the o-ring off every time it's installed. Trying to replace a used o-ring either leads to leaks or a pulsing stream (as part of the o-ring flutters in and out of the stream). We use to be able to take an o-ring in and out of the flow cell many times before it became useless. I've called BD and have tried making various adjustments but it seems that we still only get a few uses out of an o-ring. Does anyone have a solution? Thanks, any help is greatly appreciated. Geoff- Geoffrey Lyon Biotechnology Associate I FACS Facility Yale University School of Medicine TAC Building Rm-S613 300 Cedar Street Shipping Address: 1 Gilbert Street TAC Building Rm-S617 New Haven, CT 06519 New Haven, CT 06520-8035 (203) 785-2541 (203) 785-7949 -------------------------------------------------------------------------------- Replies: 1) A) Is there not surfactant in the BD approved sheath fluid and especially the one they sell for the FACS Array and cells survive. B) I agree if I remove nozzle O-ring is shredded. No solution however 2) Don't know about the surfactant issue, but as to the O-rings ... Like you I also have an Aria with an amber flow cell. I have also seen "shearing" or cutting of the O-rings under the microscope, but only rarely. Under the microscope the O-ring will have a slice or just a small chunk or pit cut out of it. When I do see O-ring problems, I also have stream problems (general instability, unusual breakoff, non-merging satellites, etc). Generally I use an O-ring for a month or more, putting it in and out a number of times each week. I often use the same O-ring on different nozzles, switching several times. I probably go through about 5-8 O-rings a year. I have also found that the O-rings are somewhat fragile. I often use a pair of very fine forceps to handle the O-rings, and I've noticed that if I grip the O-ring too tightly with the forceps I can take a chunk out of it pretty easily. I suspect you have a rough edge somewhere that's doing the damage. Perhaps either on the flow cell or on the nozzle itself. Good Luck!! 3) I have the amber flow cell and do not have the same experience. More often I lose the oring than have to replace it. In my hands, it is not a single use item. Perhaps there is an imperfection in the flow cell itself. 4) Hello, with regard to the LSR II we use sheath fluid with surfactant whether we are using the HTS module or not , we still get the odd air bubble in the system but I think this inevitable no matter what you use as a sheath fluid. I dare say it may kill the cells but I guess this will not be an issue for you if you are not sorting your cells . The question regarding the O ring for the Aria surprises me , it is our routine to remove the nozzle from the Aria every evening and "wash off" the O ring by using plastic distilled water bottle to gently concentrate a small stream of water onto the ring , washing it into a small petri dish, we then place the lid onto the petri dish until the next morning when we pick the O ring up with a disposable 100ul pipette tip and place the ring onto whichever nozzle we require , it is not unusual for us to use the same O ring for a month or longer using this daily process of removal. if you are leaving parts of the O ring behind it sounds to me as if you are leaving a nozzle in the Aria for long periods and removing it when it has partialy dried out allowing sheath crystals to form arround the nozzle and ring so when the nozzle is removed you simply pull the ring apart due to the adhesive effects of the salt crystals .If you state this happens after a single time , ie one days use , then I think you need to contact those nice people at BD 5) We've seen the same thing on our o-rings, always a little chunk the same size and presumably same location getting nicked out-- I assumed it was something with our particular flow cell, we're getting a new one Friday I'll be curious to see if it improves... didn't happen on our original clear flow cell, or on our first amber-colored one, but this is our 3rd (4th?) flow cell and we've gone through many more o-rings since its been installed... no clue about your surfactant question, so I won't even hazard a guess... 6) We have had the exact same problem with our o-rings on the Aria as well. It appears to me that it might be more the o-ring manufacturer than the flow cell. We have some o-rings that are one time use and other that we use for weeks, taking the nozzle in and out for clogs and they don't seem to chip or break at all. We've also noticed that a lot of the o-rings seem to chip in the exact same spot. Our field service engineer seems to think that it's a sharp spot on the flow cell and he is getting us a new one. Hopefully this will fix our problem, although I am still leaning towards it being a problem with the o-ring manufacturer rather than the flow cell, since we've had an amber flow cell for a while and this is a relatively new problem for us. If you find anyone with a solution, we'd love to know as well. As far as residual air floating around, once a month we run bleach for 30 mins, then detergent for 30 mins from the sheath tank through the entire system to clean it out. Then we do 30 mins of water followed by 15 mins of sheath to replace all of the water. This may help remove the bubbles and prevent you from having the risk of detergent in your sheath fluid killing your cells. Bubbles also seems to get trapped in the various connectors and valves hidden inside the cytometer itself. Taking of the bottom panel and knocking around all the connectors and valves below the surface seems to get rid of any stubborn bubbles we have that manage to make it past the bubble filter and bleed lines that we have. 7) In reply to your second question- I operate the Aria and I even pitch the o-rings that still look good if I don't like the way the stream is behaving!! They are cheap enough that it isn't worth your time to try to mess with them, and the flaws in them may be too small to see. 8) I use an Aria, we have had the amber flow cell for several years. I find O-rings often last for many (five or more) nozzle changesovers... when I an lucky. Occasionally they get lost, they have an amazing ability to jump into some hidden space and only rarely returning. So perhaps you are correct in suggesting your flow cell is especially sharp. 9) Quick answer to you question re:Aia o-rings. Do you have the new Aria 2 style flow cell? If so, why do you insist on taking it out each time? If you need to change the nozzle e.g. swapping from the closed-loop nozzle to the normal kind, and the o-ring stays inside the flow cell, just leave it there and put in the new nozzle. You should find that it sits in the right place and give you a lovely stream. I agree that the if you do need to get it out it is a bit of a pain, and I have lost more that way than with the old Aria flow cell. However, I think the new flow cell is much better and wouldn't want to go back! 10) With regard to your Purdue posting and Aria o-rings I change from the 70um nozzle to the 130um nozzle daily and sometimes get 2 exchanges before the o-ring is unsuitable and sometimes as many a 6 exchanges. I do try to pat down or seat the o-ring a little tighter by dabbing it with a lint free tissue, not sure if it makes any difference. I purchase them directly from the supplier (below). The lot I purchased last year (also supplied to BD) had 30% defective but they have a new lot now. I would have to go through a couple before one would work. Apple Rubber Products, Inc. (716) 684-6560 310 Erie street Lancaster, NY 14086 PN - R00028-008-70SLR .028 X .008 70 silicone Cost ~$9.90/ea Sincere regards, 11) We too have had some issues with the o-rings, not leaset when we recieved a batch which all contained a groove in them. We have found that our o-rings last for random periods of time, we sometimes remove the nozzle overnight, but frequesntly leave it in place, and the o-ring can be fine after being sat on the nozzle for a few days in the machine. Generally if we see leaking around our flow cell, it is the o-ring we change as that is generally the problematic piece. Wedo see a groove underneath all the nozzles which have been inserted into our amber flow cell. We have a new flow cell ready to replace the one we have, however even though we see this groove we aren't seeing any issues with seeping around the flow cell. Not sure if this really helps you in any way, but I would be really interested in what replies you have recieved as we have been informed by BD that we order an unusually high number of o-rings!Received on Mon May 12 15:18:00 2008
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