I agree the abort rate should be around 10% when running a sample at
these trigger rates on BDIS sorters. There are a couple of things that
come to mind.
Is the sample a good single cell suspension at the correct concentration?
Most people run hematopoetic cell suspensions at around 1x10^7 per mL.
Some concentrate cell suspensions even more. This will allow the sample
to run fairly quickly at low sample differential pressures, which will
usually provide a good (narrow) core stream. A lot of dead cells can
promote cell aggregates and create a more viscous suspension that seems
more prone to clog. Keeping your sample cold can improve viability as
well. You may want to review the recent threads on this list which
discuss DNAse treatment of your sample as well.
Have you checked the "dead time" or "interrogation time"? These terms
refer to the "Dead Time Adjust" knob located on card 13 on the Vantage.
You'll want to minimize the dead time so the instrument can get ready for
the next event. Turn this knob counterclockwise to shorten the dead time
while looking at pulses on the oscilloscope display. Be sure not to
shorten this too much, the instrument needs to"see" the entire pulse from
start to finish. On the Vantage, each one centimeter square represents 10
usec; our dead time is usually a little less than this. Make sure you use
your cell sample to do this; test pulses have a significantly longer
pulse width than cells. You'll notice the abort rate increase if you
shorten the dead time adjust too much.
How about the laser focus. You'll want the beam focused to a small spot
on the stream. Again, this allows for narrow pulse widths, which lead to
shorter dead times. Use the "excitation beam focus" thimble on the front
of the Vantage, and try to maximize pulse height while minimizing pulse
width.
I'm not sure if Normal-R 3 drop sorts have an effect on the abort rate.
It's not clear to me if the Sort Mode conflicts actually show up in the
system abort counters. Perhaps someone else on the list can comment on
this. Either way, most users sort in Normal-R and get abort rates around
10% of the trigger rate at low pressures and speeds.
Good luck and keep trying, it sounds like you're pretty close already.
It's fun when you finally get the instrument performing maximally.
Remember too that with cell sorting, there's no substitute for
persistence!
Hank
---------------- Begin Forwarded Message ----------------
Date: 10/23 9:06 PM
Received: 10/24 10:55 AM
From: DoranC@aol.com
To: cyto-inbox
I have recently been trained on the FACS Vantage and was told that while
sorting cells, the abort rate should not be greater than 10% of the event
rate (sys. threshold). My problem is that the abort rate is running
approx.
20 - 30%. I acquire events usually around 2000, and most often sort
normal-R, 3 drops. I have tried to alter the concentration, slow down the
rate, and even filter the sample thru a filter cap. Any ideas?
----------------- End Forwarded Message -----------------
Hank Pletcher, Technical Director
University of Pennsylvania
Cancer Center Flow Cytometry and Cell Sorter Facility
297 John Morgan Bldg/6082, 3620 Hamilton Walk
Philadelphia, PA 19104-6082
Voice: 215-898-3528 FAX: 215-898-4227