We have had similar experiences as Dr. Roederer's. I believe we have the same exceptional BD FSE helping us with our systems. I would also highly recommend the time gating strategy to correct for pressure imbalances or air bubbles on HTS systems. Steve Benoit, a colleague of mine while I was at Wyeth, effectively used this strategy on our Cyan as quality assurance for long acquisitions (i.e. rare cell detection). It would have been nice if we had a plate reading system for those long experiments on the Cyan BTW. To guard against any potential air bubble problems with mixing and sampling, we recommend that our users adjust the mixing volume to be the same as their sampling volume. We generally also like to keep the sample volume as low as is needed to achieve a minimal cell count per sample (usually 30000 in our case). A low sample volume reduces the chance of running out of sample and "sucking air". We have seen this to cause a delay followed by a sample burst at the beginning of a run. I don't know what the minimal residual volume needs to be on these systems, but I have not had problems leaving at least 25uL in well of u-bottom plates. Having not been to the LSRII class at BD, I don't know if they teach the same advice, but it helps us. Speaking of plates, we have not had trouble with any of the main plate makes. Corning/Costar, Falcon, and Nunc flat and u-bottom plates all work fine for us. As for filtration, Joanne Lannigan tipped us off to Sefar America which manufacturers and sells sheets of nylon membranes with a variety of pore size. We use these for quick filtering of a 96 well plate, with a multi-channel pipettor. We do not run tubes on our machines any more. For the past year we, at MedImmune, have used the HTS exclusively, except for Ca++ measurements, on both of our LSRII's. The HTS is a GREAT leap forward from the old MAS system. We can run a plate on average in around 30-45 minutes for immunophenotyping. We use standard mode and very rarely need to resort to HTS mode. The fluid control on these systems is fantastic, such that we routinely run 7AAD/BrdU with reasonable <5% CV's (which is very good for 7AAD). In all fairness however, we have had troubles with the reliability of these systems. In our busy lab, between 2 LSRII's, we have gone through 8 HTS systems this year. Yes, EIGHT. This is one of those cases where our service contract has been invaluable. BD has been working, so I'm told, on the problems that we have been experiencing concerning the durability of the syringe pumps on the HTS. I hope BD will announce and provide a solution soon. I also hope that other manufacturers realize the need we end users have for reasonably-automated systems so that more alternatives are available. If anyone out there wants a alpha/beta site, let me know. Also of mention, the Guava Technnologies EasyCyte has an elegantly designed plate sampling system that is very easy to use. If you or your users could use a 3, now 4-color, system I would encourage them to evaluate it for their needs. Ours is used quite frequently, also exclusively for plates. I'm eager to hear of anyone's tips or tricks on HTS use. Likewise, if anyone would like more info on sample prep or filtering info, please contact me offline. Happy Holidays, Cheers, and Happy Flowing! Chris Chris Groves Scientist I Autoimmunity and Respiratory Diseases MedImmune Inc. One MedImmune Way Gaithersburg, MD 20878 301-398-5586 grovesc@medimmune.com -----Original Message----- From: Mario Roederer [mailto:roederer@drmr.com] Sent: Mon 12/11/2006 2:08 PM To: cyto-inbox Subject: Re: BD HTS for LSRII/Canto This should be a red flag that there is a problem with your particular setup. We have had great luck with the HTS system... however, we have noticed that there is an issue in certain circumstances where the pressures are not quite balanced during the first few seconds of acquisition. This causes the stream velocity to be faster until it equilibrates. The way to diagnose this is to look at a graphic of a fluorescence parameter vs. time (or event number) -- if you see a significant change with time, you will need to gate out the initial few seconds of data. The problem is only evident on multi-laser systems (since it is an effect on the timing), and is most obvious on the laser that is furthest from the first. Also, the problem will be significantly exacerbated by using a small window extension (say, only 3); we typically run with a window extension of 10. (BTW, this is an issue on the LSR II; we have not seen it on the Calibur). The BD instrument reps have been going over our system to trace the problem; they have identified a couple of possibilities and are working hard to correct or accommodate the issue. By the way, I highly recommend viewing a graph of various fluorescence parameters vs time (or event #) on a routine basis to make sure that there are no clogs, or other issues that can induce variabilities in the fluorescence that can cause artefacts and "bad data." In any case, we have 5 HTS systems, and they perform very well. Indeed, when we first installed an HTS unit, we discovered that our users wouldn't use the non-HTS-equipped system, so we were "forced" to buy HTS on all the machines... mr On Dec 8, 2006, at 2:04 PM, Watson, Susan wrote: > We got one to use for screening mouse blood. > After 3 months we gave up using it cos there was too > much variation in the data as compared with hand running the samples. > SRW > > > -----Original Message----- > From: Adrian Smith [mailto:a.smith@centenary.usyd.edu.au] > Sent: Thu 12/7/2006 5:44 PM > To: cyto-inbox > Subject: BD HTS for LSRII/Canto > Hi all, > > We are considering adding a 96-welll plate reader to LSRII or Canto > and I'm putting together a grant application for the BD HTS. > > I'm looking for feedback from current users on reliablity and ease of > use. > > I'm also particularly interested in what people are using it for? > What sort of efficiency improvements does it provide over tubes in > non-highthroughput scenario (ie most of what we do requires > reasonably large files). > > I know I want one - I just need some convincing arguments for the > funding committee :) > > Regards, > > Adrian Smith > Centenary Institute, Sydney, Australia > > >Received on Mon Dec 18 16:38:00 2006
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