Darlene Steele-Norwood asked about setting up the FacStar+ for 4-color flow using FITC/PE/Tricolor/APC with argon and HeNe lasers and a 650LP filter. --------------------------------------- We have not tried this exact combination, but have tried detecting 7-AAD in FL3 from the argon laser. You have to be careful to set up the instrument for the correct adjustment of the dichoic mirrors that deflect the two red signals. This means adjusting the vertical setting of the 90 degree optics while watching the signal. If this is off, you get no FL3 from the argon laser. It is probably best to use a particle that fluorescences red from both of the lasers (fixed chicken cells work as do DNA check beads from Coulter and Rainbow beads from Spherotech), but other beads or cells may also work that are closer to the characteristics of your cells. They would need to be mixed in approximately equal amounts. Once you have this adjusted you should be able to detect your FL3 signal, unless it is dim. The FACStar+ is not as sensitive as the FACScan, so your dot patterns will appear dimmer. You may need to select the brightest signal for FL3. Good luck, Tony Bakke, PhD Director, Flow Cytometry Lab Oregon Health Sciences Univ.
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