Fluorescence instability of propidium iodide‐labelled microspheres in (para)formaldehyde

Abstract

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. © 1993 Wiley‐Liss, Inc. Copyright © 1993 Wiley‐Liss, Inc.