Rapid and simple kinetic cytolysis assay of toxin by Flow Cytometry.

Background: Cell viability assays by using specific dyes such as florescence and radioactive materials are a necessary technique in the research laboratory. However, these methods are not suitable for cytolytic activity of membrane-damage toxins. Therefore, a new method to evaluate cytolytic activity is requisite for researchers. The aim of this study was to evaluate a flow cytometry for cell lysis assay of pore-forming toxin, a membrane-damage toxin.  Materials and Methods: we used two light-scatter factors of flow cytometry (Side-Scattered for cell size and Forward-Scattered for cell granularity) to quantitatively analyze cell lysis by pore-forming toxin, alpha-hemolysin (αHL). To verify efficiency of our approach, we compared the results from flow cytometric cytolysis assay with that from conventional cell lysis assay with microplate reader.  Results:  We successfully assessed the cytolytic activity of αHL toward not only rabbit red blood cells but also cultured human cells with flow cytometry in time and concentration-dependent manner:  Times to 50 % rabbit red blood cell lysis were 3 min, 6 min, and 9 min by 20 ul, 10 ul, and 5 ul of 0.5 nM alpha hemolysin respectively; times to 50 % human leukemia cells lysis were 25 min, 37 min, and 55 min by  40 ul, 20 ul, and 10 ul of alpha hemolysin respectively.  Conclusion: We conclude that our approach, kinetic cytolysis assay using flow cytometry, allows researchers to simply monitor cell-mediated cytotolytic activity of membrane-damage toxin.