Hi Derek,
This has been a source of annoyance to me too, I find that if I get people
to run the buffer/medium that they've suspended their cells in (she doesn't
use distilled water does she?), the "problem" goes away, if it doesn't I
suggest filtering it. You can tell if it's a problem with the fluidics by
seeing what happens when you take the tube off in run, if the particles
still show up (before the laser powers down) then it's probably a sheath
problem, if they stop flowing before the standby click, it's probably in
the water. Most sources of dH20 have a tank that grows algae, autoclaving
doesn't get rid of them, give her a stiff talking to about appropriate
controls:-)
Ray
At 7:59 +0000 2/12/97, Derek Davies wrote:
>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 *
>****************************************************************************
Ray Hicks
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