Fluorescence calibration

Hans-Peter Spengler hspengl at gwdg.de
Wed Feb 26 15:01:02 EST 1997


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. 



Hans-Peter Spengler, PhD
Dept. of Immunology                  FAX:   +49-551-395843
University of Goettingen             Phone: +49-551-395819
Kreuzbergring 57                     E-mail: hspengl at gwdg.de  
D-37075 Goettingen/Germany

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