Hi Glenn,
You ought to get the same results - one point is that since the sample size is
high you need not count. You can just compare the -1 drop and +1 drop puddles
until they contain the same number of outliers: speed. Counter mode has the extra
0.5 drop on each side, so it requires more settings to determine the delay. If you
use Normal-R, there is no extra time added to the envelope, so 1 drop packets are
reliable. I never said you would get different results, only that using Normal-R
and the 3 puddle method is faster because the sample size is huge and the timing
is 1.0 drop at 1 drop. I said "may be asking for trouble" using a 1.3 drop
deflection envelope after a traditional Counter method. We usually time the sorter
in couple of minutes, the traditional drop delay profile takes a lot longer. My
other point, accurate means a sample size of 1500 +/- 100 is easier to spot with 1
drop envelopes than 50 +/- 1 with 1.5 drop envelopes, doesn't mean you'll get a
wrong result - only that you'll need more deflection samples with different time
increments to determine the delay.
Joe
Glenn Paradis wrote:
> Hi Joe,
>
> I use counter mode to set drop delay all the time. I sort 50 beads at the
> different delays and see which ones give me the best recovery. I heard
> that you said that drop delays in counter vs normal-r would give different
> results so I tested them and got exactly the same drop profile in both
> modes. I did this on a TSO Vantage and my FACStar Plus.
>
> Just wondering about this normal-r vs counter mode difference??????
>
> Glenn
>
> *************************************************
> Glenn Paradis
> MIT Flow Cytometry Core Facility
> E18-463
> Massachusetts Institute of Technology
> Cambridge, MA 02139
> (617)253-6454 lab
> (617)452-4132 office
> gap@mit.edu
> *************************************************
>
> >Doing a drop delay profile on a Vantage using Counter mode and then sorting
> >in Normal-R mode with a 1.3 drop deflection envelope may be asking for
> >trouble. Counter mode adds 0.5 drops on either side of the drop envelope,
> >thereby masking the prescision one needs to be able to clock down to a 1.3
> >drop window. I would suggest using the "3 puddle" method where you sort in
> >Normal-R using 1.0 drop envelopes at: -1 drop, 0 drop, +1 drop from the best
> >estimate of the delay running particles at ~ 500/second. Sort 1000 - 1500
> >events into each puddle and adjust the delay such that you get the same
> >number of outliers in the -1 and +1 offsets (and the vast majority in the
> >middle puddle). By using a real 1.0 drop envelope and using the larger sample
> >size it is far easier to accurately time the instrument to within 0.1 drops.
> >We have never used traditional drop delay profiles to time the sorters since
> >the "3 puddle" method is faster and more accurate.
> >
> >The Vantage SE is a essentially a digital sort system, whereas the Vantage
> >uses faster peak sense and hold circuits inherited from the FACStar Plus.
> >Upgrading a Vantage to a Vantage SE makes a lot of sense if you want better
> >sorting at "TurboSort" performance.
> >
> >
> > Regards,
> >
> > Joe
> >
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