time delay calibration particle

Simon Monard smonard at trudeauinstitute.org
Wed Oct 4 09:37:20 EST 2000

I use the 6u Alignflow beads with no problems.

Simon Monard
FACS Lab Manager
Trudeau Institute
Saranac Lake

Ph 518 891 3080 X352

>>> Glenn Paradis <gap at MIT.EDU> - 10/3/2000 5:00 PM >>>

Hi Everyone,

I have a new twist to the problem of an improper time delay calibration on
our FACS Calibur.  I have isolated the problem to the beads I am using to
perform the time delay calibration and am wondering if someone has an
explanation for my observation.

I have been using an old lot of 6.0 um Polyscience beads which is now
running out.  The time delay calibration has been and still is working
flawlessly with the old Polyscience beads.  I recently purchased 633nm
excitation, 2.5 um Molecular Probes AlignFlow beads A-7312.  The beads
excite very well with the diode laser and are much brighter the the
Polyscience beads.  After increasing the FSC gain to put the beads in
channel 400 and decreasing the Fl4 PMT voltage to put the beads in the
third log decade I get an error saying the event rate is too low for the
Fl4 fluorescence.  By looking at the histograms, there are clearly lots of
beads running through the cytometer and the counters say that the number of
beads is 1,500/sec.

When I open the FACS Calibur hood I see the yellow light on the time delay
board is shining brightly.  I have tried running in lo, med, and hi with no
successful calibration.  I have tried resetting the time delay switches on
the board and nothing seems to help.  As soon as I put my old beads back on
with my normal settings, I get a nice happy beep telling  me my time delay
calibration has worked.

Is there a minimum size particle that must be used in order to time delay
calibrate?  Any ideas would be appreciated.



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