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 Davies Voice: (44) 0171 269 3394 * * FACS Laboratory, FAX: (44) 0171 269 3100 * * Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk * * London, UK * * * * Web Page: http://www.icnet.uk/axp/facs/davies/index.html * ****************************************************************************
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