Crossbeam compensation on the FACSCalibur.
Our FACSCalibur has been optimized for simultaneous use of PE-Cy5 and APC with minimal crossbeam compensation, according to the procedures worked out by Barry Mallow at Becton-Dickinson Biosciences and Kevin Holmes of the NIAID Flow Laboratory. Diode laser excitation of PE-Cy5 can generally be compensated out of the FL4 PMT (APC or Cy5) with less than 40% compensation.
(Below). EL4 thymoma cells were labeled with biotin-anti-Thy1.2 followed by either PE-Cy5-avidin, APC-avidin, or Cy5-avidin. Cell mixtures of unlabeled, PE-Cy5- and APC-labeled cells were then analyzed on the FACSCalibur with and without crossbeam compensation. Compensation levels were FL3-%FL4 = 3.3% and FL4-%FL3 = 34.7%.
(Below). Cell mixtures of unlabeled, PE-Cy5- and Cy5-labeled cells were analyzed on the FACSCalibur with and without crossbeam compensation. Compensation levels were the same as above.
