[Cytometry] Problem with BD CS&T laser settings

DAGNA S SHEERAR dsheerar at wisc.edu
Fri Jul 24 14:06:41 EDT 2009

Hello Everyone - 

I'm not entirely sure I agree with using the CST settings for experiments.  I do however heartily agree with the statement that we need to get used to looking at our negative populations higher up the scale.  There are several "dim" fluorochromes that necessitate this.  Such as Pacific Blue, Pacific Orange, Alexa 700, V450...  When there is less than a 1-2 log shift between "negative" and "positive" populations, it is very helpful to set voltages on these PMTs such that the negative is around the top of the first decade or higher.  

In terms of standardizing an experiment, it might be better to use a bead to set your voltages.  By this I mean, run your CST, set your PMTs based on your unstained control and then run a bead like Spherotech's Rainbow.  Note the means in each of your PMTs and then use that as your standard each time you run that specific experiment.  That way, not only will your data look good from run to run, but you should be able to compare fluorescent means from day to day, week to week, etc.  I don't know enough about the CST to say if this would work based on those settings, as machines do fluctuate from day to day, as we all know.  Using a specific bead standard based on means for each specific experiment would eliminate error due to adjusting those dim or bright populations by eye.

I have to come to think of CST as a measure of instrument performance, much like FACSComp on the Calibur.  We don't run all our experiments on the Calibur with the FACSComp settings; I fail to see why we'd do it on the LSR or DiVa.

just my two cents...

Dagna Sheerar
University of Wisconsin - Madison
Carbone Comprehensive Cancer Center
Flow Cytometry Facility
1111 Highland Ave
7016 WIMR
Madison, WI 53706
dsheerar at wisc.edu

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