FLUORESCENCE INSTABILITY OF PROPIDIUM IODIDE-LABELED MICROSPHERES IN (PARA)FORMALDEHYDE

An instability of the fluorescence of propidium iodide (PI)-labelled m icrospheres was observed when the beads were used as an internal fluor escence standard in cell samples suspended in paraformaldehyde (pFA) f or flow cytometric phenotyping. Flow cytometry of fluorochrome-labelle d microspheres as well as spectrofluorometry of PI-fluorochrome soluti ons in PBS-buffered 1% pFA or formalin (FA) revealed strong increases of PI fluorescence starting immediately after the addition to the fixa tives. We propose a chemical reaction which leads to two additional co njugated double bonds in the modified fluorochrome causing the increas ed fluorescence emission. Therefore, PI-labelled microspheres should n ot be applied as internal fluorescence references in aldehyde-containi ng cell suspensions for flow cytometric analysis, except when the time interval between the addition of beads and the measurement can be kep t constant or if it lasts longer than 1 h.