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Fluorescence instability of propidium iodide-labelled microspheres in (para)formaldehyde
1Institute of Radiobiology, Federal Armed Forces Medical Academy, Munich, Germany.
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.
Insights
Propidium iodide (PI) fluorescence instability in microspheres used as standards is caused by aldehyde fixatives. Avoid using PI-labeled microspheres in aldehyde-containing samples for flow cytometry unless measurements are immediate or delayed by over an hour.
Area of Science:
- Flow cytometry
- Fluorescence spectroscopy
- Biochemistry
Background:
- Microspheres are commonly used as internal standards in flow cytometry for accurate fluorescence quantification.
- Propidium iodide (PI) is a vital stain and a common fluorochrome used in flow cytometry.
- Aldehyde fixatives like paraformaldehyde (pFA) and formalin (FA) are widely used to preserve cellular structures.
Purpose of the Study:
- To investigate the observed instability of propidium iodide (PI)-labeled microspheres when used as internal fluorescence standards.
- To determine the cause of PI fluorescence enhancement in the presence of aldehyde fixatives.
- To provide recommendations for the appropriate use of PI-labeled microspheres in flow cytometry.
Main Methods:
- Flow cytometry analysis of PI-labeled microspheres in paraformaldehyde (pFA) and formalin (FA) fixed cell suspensions.
- Spectrofluorometry of PI-fluorochrome solutions in phosphate-buffered saline (PBS) with pFA or FA.
- Chemical analysis to elucidate the mechanism of PI fluorescence enhancement.
Main Results:
- PI-labeled microspheres exhibited significant fluorescence instability when suspended in aldehyde fixatives (pFA, FA).
- A marked increase in PI fluorescence was observed immediately upon addition to pFA or FA solutions.
- Spectrofluorometry confirmed enhanced PI fluorescence in the presence of aldehydes, suggesting a chemical modification of the fluorochrome.
Conclusions:
- PI-labeled microspheres are unsuitable as internal fluorescence standards in aldehyde-containing cell suspensions for flow cytometry.
- Aldehyde fixatives induce a chemical reaction that increases PI fluorescence, likely through the formation of additional conjugated double bonds.
- The use of PI-labeled microspheres as internal standards is permissible only if the time between bead addition and measurement is constant or exceeds 1 hour.