Dear flow-community, I am trying to sort cancer cell line side population (SP) on BD Aria equipped with 50 mW 405 laser. Our initial studies were looking at bone marrow SP with Hoechst33342 and Invitrogen Dye Cycle Violet (DCV) on our LSR II (404 25 mW laser; 670LP, 450/50). Both gave reasonably good resolution of a verapamil sensitive SP. Same was true for looking at a number of cancer cell lines. The problem is that when trying to sort this population on the Aria it is hard for me to find a gate that would have very few cells in the verapamil treated samples but similar to LSR II % positive in the non-treated samples. DCV gave a bit better picture than Hoechst, but is still a far cry from ideal. Did anyone successfully sort SP on Aria with violet laser? Any suggestions? Side questions: why would a stronger laser on Aria give poorer resolution than a weaker on the LSR - is it the beam width? Would running in "Low sort" instead of "High Sort" increase the illumination time and give a better result? Thank you, Charlie USF Tampa PS on the attached figures you may point out that on the Aria I used less blue voltage, but applying more does not raise the non-SP be horizontally, it would only be going across the plot in a diagonal more. This attachment - 'SP question.doc' - 420.35 KBytes - can be viewed at http://www.cyto.purdue.edu/MD-parts/fcfc855fd78c918b5c26ceeb57dbff85e608c460.docReceived on Wed May 7 14:38:00 2008
This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 - 03:12:00 EST
