Abstract
PurposeThe aim of this work was to develop a quantitative, flow cytometric method for tracking the endolysosomal escape of a fluorescently labelled saporin toxin.MethodsFlow cytometric measurements of fluorescent pulse width and height were used to track the endocytic uptake into Daudi cells of a fluorescently labelled saporin toxin and the saporin based immunotoxin, OKT10-SAP. Subsequently, measurement of changes in pulse width were used to investigate the effect of a triterpenoid saponin on the endolysosomal escape of internalised toxin into the cytosol. Live cell confocal microscopy was used to validate the flow cytometry data.ResultsIncreased endolysosomal escape of saporin and OKT10-SAP was observed by confocal microscopy in cells treated with saponin. Fluorescent pulse width measurements were also able to detect and quantify escape more sensitively than confocal microscopy. Saponin induced endolysosomal escape could be abrogated by treatment with chloroquine, an inhibitor of endolysosomal acidification. Chloroquine abrogation of escape was also mirrored by a concomitant abrogation of cytotoxicity.ConclusionsPoor endolysosomal escape is often a rate limiting step for the cytosolic delivery of protein toxins and other macromolecules. Pulse width analysis offers a simple method to semi-quantify the endolysosomal escape of this and similar molecules into the cytosol.
Highlights
The delivery of macromolecules, such as protein therapeutics or nucleic acids, into cells often depends on endocytic processes
We present here a flow cytometric assay to measure the endolysosomal escape of the type I ribosome inactivating protein saporin, a key component of many targeted toxins [2,3,4]
In this study we demonstrate the use of pulse width analysis as a tool to investigate the enhancement of the endolysosomal escape of saporin and the saporin based immunotoxin (IT) OKT10-SAP by triterpenoid saponins from Gypsophila species
Summary
The delivery of macromolecules, such as protein therapeutics or nucleic acids, into cells often depends on endocytic processes. Following internalisation into the early endosome, the compartment matures into the late endosome before fusing with lysosomes to form the endo-lysosome where the contents are degraded by lysosomal enzymes [1]. For these macromolecules to reach their targets in the cytosol, they must escape the endolysosomal compartment before they are degraded. We present here a flow cytometric assay to measure the endolysosomal escape of the type I ribosome inactivating protein saporin, a key component of many targeted toxins [2,3,4]
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