4-color flow -Reply

Antony Bakke (bakkea@ohsu.edu)
Mon, 23 Jun 1997 16:41:45 -0700

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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