Increasing the flow rate increases the frequency of doublets (2 cells) being detected as single events and thus yielding false double positive cells. A couple of years back we tried running our FACSCalibur at rates over 3,000 events per second in order acquire sufficient number of events to detect Ag specific cytokine responses. in a pilot, we stained separate samples for CD3 FITC and CD3 PE and then mixed the cells together after staining. Gating on lymphocytes by scatter reduced the number of double positive cells, since most of the doublets had a greater forward scatter. Even though we gated on the lymphocyte cluster, as the rate increased, so did frequency of double positive events. For the events we were studying, we found that 3,000 events was the maximum event rate we could use and still be assured that our double positive cells were actually not 2 signal positive cells triggering at the same time. Calman > ---------- > From: Rosson, Dan > Sent: Thursday, June 14, 2001 7:54 > To: Cytometry Mailing List > Subject: Flow rates and the effect on data > > > I have what for some of you will be an elementary question. I've asked 3 > people who've been in the business 20 times longer than I have and are in > a > position to know, and I've gotten 2 different answers. > > A FACScan has a limit as to how many cells it can count per second. If > the > cells are going through the flow cell faster than the reset time allows it > to count, what happens to your data? > Does the FACscan simply "ignore" a percentage of the cells, in which case > one doesn't really care because if the sample is that concentrated you > have > more than enough cells to do your analysis? Or, does the instrument see > partial signals as the next cell arrives in the flow cell before the > instrument has had time to reset itself, thus giving rise to lower MFI's > and > wider CV's, in which case one does care? > > A search of the e-mail archives uncovered related discussions that imply > to > me that the former explanation (it ignores some cells) is the answer, but > I'd like some reassurance. > > > > > > Dan Rosson Ph.D. > Lankenau Medical Research Center > >
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:57:44 EST