Hazel, we use the DAKO FluoroSpheres but for daily monitoring of the flow cytometer (FACSstar Plus) settings we traditionally use the Calibrite Beads Kit purchased from Beckton Dickinson. The kit is also very usefull when the compensation settings have to be check for the FITC and PE channel. Because of their higher forward and side scatter light compared to the DAKO FluoroSpheres and the similarities to lymphocytes it is easy to detect an improper flow. The Calibrite Beads Kit consist of three dropper bottles, one with unlabeled and the others with FITC-conjugated and PE-conjugated beads. For a working batch we mix one drop from each bottle in approx. 5 ml sheet fluid and store it in the refrigerator until use. Additionally, when we try to compare results obtained from the same experiments but performed on different days then we use the DAKO FluoroSpheres (40 tests). They enable the transformation of arbitrary units (e.g. channel numbers or mean fluorescence intensities) into absolute units by use of a calculation programm. The DAKO leaflet describes microspheres of six different fluorescence intensities. In fact, the kit contains two dropper bottles ready to use, vial no.1 with the blank beads and vial no.2 with a mixture of the 3.2 micron fluorescent beads of five different fluorescence intensities excited at any wavelength from 364 to 650 nm. At the beginning of a FACS analysis we run first the blank beads and then the fluorescence beads followed by cell analysis. At the end it is recommendable to re-analyze the DAKO beads again. For calculation DAKO offers TallyCAL, a Windows-based software programm (Code No. S2033, approx. DM1200, including one kit FluoroSpheres) which encompasses several protocols for different flow cytometers and their specific analysis software. To our surprise, calculation done with MS-Excel showed differences (approx. 2 per cent) with the results calculated with TallyCAL. In conclusion, I like both the FluoroSpheres beads and the TallyCal programm. Using both you will get comparable results even in experiments where receptor regulation will be investigated, and don't worry about day to day variations of the flow cytometer settings, really a problem when using a FACStar. Cheers, HP -------------------------------------------------------------------------- Hans-Peter Spengler, PhD Dept. of Immunology FAX: +49-551-395843 University of Goettingen Phone: +49-551-395819 Kreuzbergring 57 E-mail: hspengl@gwdg.de D-37075 Goettingen/Germany
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