I will post a hypothesis on why the SSC decreases with increased flow rate when there is a significant number of particles below threshold (FSC). The DiVa systems (Aria, Canto, VantageDiVa) have a base line subtraction calculation whereby each measurement channel is sampled between threshold events and an average value calculated which is then subtracted from the measurements on that channel (when measuring Area). So if you had s lot of RBCs in your sample below threshold, the faster you ran your sample, the higher your average background baseline on SSC will be. To see if this is the case, you could look at SSC-H. This does not have an integrated baseline subtracted, so it should remain constant regardless of flow rate. Marty Marty Bigos Director, Flow Cytometry Core Laboratory Gladstone Institute of Virology and Immunology Mail & Deliveries: 1650 Owens Street Rm 521 San Francisco CA 94158 (tel) 415-734-4821 (fax) 415-355-0855 (mobile) 415-845-8450 mbigos@gladstone.ucsf.edu http://www.gladstone.ucsf.edu http://www.gladstone.ucsf.edu/gladstone/php/?sitename=flowcytometry >On 24/05/2006, at 10:13 AM, PIETRO BULIAN wrote: > >>Hello All, >> >>Could high flow rate of unobserved cells (under FSC threshold), >>like RBC, affect side scatter measurements, inducing a decrease in >>signal? I was looking at lymphocytes in a PB sample (lambda-FITC, >>kappa-PE, CD19-PecP-Cy5.5, CD10-APC) with incomplete RBC lysis on a >>BD Canto. I recorded 30.000 events with threshold on FSC at low, >>medium and high flow rate in 109, 9 and 7 seconds respectively >>which equals 275, 3333, 4286 cells/sec (see the attached picture). >>Unfortunately I did not estimate the RBC concentration (at visual >>inspection it was rather high), and so I don't know the total >>particle concentration. But RBC under and even above threshold >>should not be a problem: in a previous tread ("going too fast") >>about increasing sorting rate using a fluorescence threshold, a >>comment by dr Shapiro reported about analyzing leukocytes in >>unlysed whole blood at 50.000 cells/second, with scatter >>measurements for sizing. BD declares 10.000 cells/sec for >>acquisition rate on Canto, in my experiments I was under this >>value. When I analyze purified leucocytes I don't see this >>phenomenon even at greater rates, so I suppose that the problem is >>high concentration of unobserved RBC, with very high total particle >>flow rate, but shouldn't the system be blind to what is under >>trigger? Aside for my 2006' Canto (and me) be unable to do what dr >>Shapiro did in mid-1970s, what is the explanation: coincidence, >>electronic? Why is so affected side scatter (the medians of forward >>scatter and FL are less decreased)? >> > > >We see this also on our FACSCanto... interestingly it only seems to >effect SSC-A not SSC-H... > >I'll also have the impression that it is related to the events below >threshold and I have some theories about why it might happen >(related to the way I think DiVa sets baselines) but I'm keen to see >what the real experts say...(ie those who actually know about the >inner >workings of the Canto/DiVa system rather than someone like me who is >relying on what I remember being said at various workshops)... > >Regards, > >Adrian Smith >Centenary Institute --Received on Mon Jun 5 15:49:49 2006
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