Fluorescence instability of propidium iodide-labelled microspheres in (para)formaldehyde.
An instability of the fluorescence of propidium iodide (PI)-labelled microspheres was observed when the beads were used as an internal fluorescence standard in cell samples suspended in paraformaldehyde (pFA) for flow cytometric phenotyping. Flow cytometry of fluorochrome-labelled microspheres as well as spectrofluorometry of PI-fluorochrome solutions in PBS-buffered 1% pFA or formalin (FA) revealed strong increases of PI fluorescence starting immediately after the addition to the fixatives. We propose a chemical reaction which leads to two additional conjugated double bonds in the modified fluorochrome causing the increased fluorescence emission. Therefore, PI-labelled microspheres should not be applied as internal fluorescence references in aldehyde-containing cell suspensions for flow cytometric analysis, except when the time interval between the addition of beads and the measurement can be kept constant or if it lasts longer than 1 h.
Rohr
E L
EL
Institute of Radiobiology, Federal Armed Forces Medical Academy, Munich, Germany.
Kaffenberger
W
W
D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D005557:Formaldehyde; D006801:Humans; D007962:Leukocytes / Q000166:cytology; D008863:Microspheres; D010641:Phenotype; D011108:Polymers; D011419:Propidium
D011108:Polymers; D005557:Formaldehyde; D011419:Propidium; C003043:paraform