Quantitative, High-Throughput, Fluorescent-Based Bioassay to Detect Schistosoma Viability
Application Note May 31, 2011
This application note presents a fluorescence intensity based microtiter plate assay to reproducibly detect schistosomal viability using the POLARstar Omega from BMG LABTECH.
The principle of the assay is based on differential membrane permeabilities of the dyes, fluorescein diacetate (FDA) and propidium iodide (PI). Fluorescein diacetate is able to cross the membrane of living cells. Once inside the cell, esterases will cut the diacetate and fluorescein is released resulting in a measurable fluorescence signal that is directly related to the number of living cells. In contrast, propidium iodide cannot enter a viable cell. This dye will only stain the DNA of dead cells when the membrane has been compromised. The simultaneous detection of both propidium iodide and fluorescein diacetate measures allowed us to develop a fluorescence-based, microplate bioassay to improve detection of schistosomal viability. The assay is adaptive to 96-well and 384-well format, quantitative and provides objective readouts (relative fluorescence units) of parasite viability during in vitro culture. Using this flexible bioassay, we demonstrate its versatility in detecting schistosomal viability in response to a known inhibitor of thioredoxin glutathione reductase (auranofin).