[Cytometry] Compensation on High Auto-fluorescent cells

Lorelle Parker lparker at chori.org
Wed Feb 26 19:08:48 EST 2014

Hello All--- 
I have a researcher that would like to sort primary tumor cells on our Aria IIu. We have a UV laser with two PMTs, a red with three, and a blue with five. 
As a requirement with all new sorts I had her bring me some unstained cells to run through to get a profile before she begins experimentation (she also wanted to know what fluorescent marker would be best to use). 
The cells were very, very auto-fluorescent, even with very tight discriminant gates. 
I opened a histogram off of each PMT, and the signal for the unstained cells had a clear negative peak around 10^2, but in some there were a mid peak around 10^3, and then all had a very clear positive peak between 10^4 and 10^5 for all of the PMTs off of both the UV, all off the Blue laser, and in the APC channel on the red. The other two channels on the Red laser were negative (just a single peak at 10^2). 
She is happy looking for a marker that we can run off the red laser. But I have to ask now, what is the best way to do compensation? 
Normally I require that everyone bring unstained cells along with the regular compensation tube, but the unstained cells are usually negative off of each laser. The sample does have a clear negative peak, but with the high signal in all of the other channels, should I run compensation in each PMT? Should I have her bring some other type of negative cell or compensation bead? She isn't really keen on treated the cells with anything to lower the auto-fluorescence. 



Lorelle S. Parker, M.S., Psy.D. 
Flow Core Facility 
Children's Hospital Oakland Research Institute 
5700 Martin Luther King Jr. Way 
Oakland, California 94609 
lparker at chori.org 

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