How do I calibrate my system for flow cytometry?
Flow cytometers seldom have an optimal wavelength for a dye of interest (except the 488 line optimized for FITC, Cy2 and Alexa 488). Thus, a proper calibration standard requires optical properties which are exactly the same as the dye being used. This becomes even more of a problem with different flow cytometers which may use different wavelengths. We have generally found that the ‘calibration beads’ sold by various manufacturers are unsatisfactory for such use. Thus, we treat an established cell line (V79 Chines Hamster Cells) with a defined exposure of EF5 and oxygen (400µM/hr, < 0.005%), fix the cells and then freeze them in 25% glycerine. This defines a standard based on cellular content of antigen. Thus, regardless of what 'color' of antibody is used, the very same antibody can be used to stain the standard cells, and we set the cytometer to read a mean fluorescence of '1000' on a 1 to 10,000 scale. As with the microscopy standard, we have found that cytometers can change their 'r
