Hello C Doran, 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
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