>Hello fellow fans of flow! > >I apologise in advance but I would like to return to the ever-popular >cytometer cleaning question. > >I have a FACScan and a FACS Calibur, both multi-user high throughput >machines, both used for DNA and for phenotyping experiments. I have >impressed upon all my users that after running their samples they MUST run >a cleaning procedure which is: > >1. dH2O 5 mins on high flow >2. detergent (7X) 5 mins high flow >3. dH2O 2 mins high flow (if PI has been used I put a 5 min bleach step >subsequent to this) >4. Standby (If I am about I tend to leave it on run) > >I also run detergent and dH2O through the entire fluidics system once a >week. So far so good, rarely get clogs, bead and DNA CVs and profiles >fine, haven't had to slay too many users for forgetting and I am happy >(well as happy as I get these days). Recently though I have been informed >of a few 'problems'. I have one user in particular who quite often ends up >on the linoleum in a black apoplexy. She is running 3 or 4 colour fetal >thymocytes. Before she starts her run she acquires 'data' from a tube of >autoclaved distilled water. Her complaint is that events are acquired that >fall within her cell gate. So.....my question is this: > >Should I expect to see 'cells' in a tube of dH2O? If so, how many, say, >per minute? I have just run such a tube on high flow rate and got over a >10 minute period an average of 2-4 events per second (Low and high flow) >in a typical cell gate. I would be tempted to say that this is >insignificant in a real sample compared with the cell flow rate. > >Does anyone have a view on what is acceptable (I checked the archives as >there was a flurry of cleaning correspondence earlier in the year but >couldnt find the answer!)? Am I wrong in saying that there would never be >a *totally* clean background? > >Oh I use PBS as sheath fluid and I have changed the in-line filter. > >Thanks in advance! > >Derek Derek, Jeez, with everything you do, how can she complain? Even if she acquired background events in her cell gates, they would be a) so few that they would be statistacally insignificant, and b) shouldn't fluoresce, escpecially if she is doing 3 and 4 colors. That is if what she is "seeing" is background from the DI. Have you shown her the data you acquired from your 10 minute acquisition of DI? By the way, how do you get your users to comply with your cleaning regime? I can barely get the users here to put water on and let it run for a minute. Another question, do you pre-fill tubes with water, detergant and bleach, or do you have squirt bottles. And do you filter the different solutions? Thanks, Bill Nostrom Trudeau Institute Saranac Lake, NY PS. Does she really end up on the linoleum in a black apoplexy? Ouch, and I suppose the janitors have to clean up the mess, eh? ______________________________________________________ Get Your Private, Free Email at http://www.hotmail.com
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