CytoFlex LX settings optimization
Hi all,
I’m new to the CytoFlex LX and would like to share my observations and get some advice from this community. My LX is equipped with the deep well plate loader module.
When running samples (single stained comps for now) using standard 96-well v bottom plate I see this strange phenomenon where signal from the previous well is contaminating the next sample but that signal gradually goes away with time (number of events go substantially down after about 10-20 seconds). I use 1.5 sec mix and 5 second backflush. I should mention that the event rate initially is also somewhat higher than later but the loss in contaminating events is still greater given the percent of contaminating events per a time interval goes down with time if that makes sense (almost ~15 % during first 15 seconds, then ~5% during next 15 seconds, then ~0.5% during the next 30 seconds at Fast Flow Rate ). Do I need to make backflush even longer?
When going from Sample Flow Rate Slow (10uL/min), to Medium (30uL/min), to Fast (60uL/min) I see very noticeable broadening of signal in pretty much every channel including scatter. Is this me or is this particularly true for this specific platform? I don’t recall this with BD instruments (Fortessa) when going between flow rates. I believe others observe this. What is your recommended sample acquisition settings then? Concentrated samples (50uL instead of 200uL per well final resuspension in plate?) acquired slow?
On a Time vs Fluorescence or Scatter gate, I see what appears to be a gap of about 1 second in data every about 10 seconds. I assume this is the peristaltic pump. I thought swapping the peristaltic sample line would alleviate this but it did not. Is this normal behavior then?
Most concerning to me is #1 as I was expecting carryover to be <0.5% according to the spec of this instrument. I appreciate everyone’s input.