Tracking settings

When a researcher is using the same panel for experiments run on different days, the temptation is to just use the same voltages as the first day.  Due to instrument variations (fluctuation in laser power, variation in detector sensitivity due to heat and humidity,  how long the instrument has been on, etc), this likely will not give you the same results.  The only way to compare samples run on different days is to make sure the instrument is providing the same output for the same fluorescence signal on both days, to confirm that differences between data files are due to actual sample differences and not to instrument variation.

Keep in mind that staining consistency is also critical, as variation in your technique can lead to differences measured on the cytometer, and a good control for this is to run the same sample on each day and make sure they are consistent– so have a bank of PBMC aliquots from the same patient, and stain one with each experiment.  If the instrument is consistent, and the sample is matched, then any variation would be due to changes in the staining and this will give you an idea of the consistency of your staining on different days, and how much of a change is actually significant.

With BD instruments, CST (Cytometer Setup and Tracking) in Diva can adjust voltages based on daily CST results (“Application Settings”), but you have to re-synchronize the settings for each new configuration, batch of CST or each new baseline for EACH user, which becomes challenging for a core facility with many users.

Another benefit of monitoring your instrument response for your experiment is that if someone has swapped filters on the instrument and neglected to restore them to “default”, it will usually be readily apparent in a large shift in your bead peak and hopefully shorten any troubleshooting you might have to do (“Gee, my pacific blue single stain has no signal, let me re-make the comp tube.  Still didn’t work, oh wait someone swapped the filter…”).

Here is the writeup:

https://newenglandcytometry.files.wordpress.com/2015/02/rainbowtracksettings.pdf

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