Improved method to evaluate the ability of compounds to destabilize the cellular plasma membrane

In the paper, we present an improved method for evaluation of a compound ability to destabilize erythrocyte plasma membrane. The proposed method is based on the continuous monitoring of the light scattered by erythrocytes exposed to osmotic pressure differences. The kinetics of hemolysis depends on the plasma membrane mechanics and the extent of the osmotic stress. Generally, the osmotic pressure difference of approximately 150 mOsm is taken for measurements, as a result of the equal volume mixing with the physiological salt solutions. In this approach the hemolytic process completion is not established which may result in poor quality and reproducibility of the experimental data. In consequence, inaccurate parameters of the kinetic are determined due to the low quality fitting to the, widely used, single exponential model. In the paper we propose a new experimental protocol allowing to determine the extended set of parameters for kinetics of hemolysis. Namely, the method of the minimal osmotic pressure difference determination is proposed which ensures the completeness of the hemolytic process. This step allows improving the quality and exactness of the calculated parameters. The developed methodology was tested on two qualitatively different, biologically relevant, experiments; evaluation of the peptide effect on the plasma membrane properties and differentiating between human and rabbit erythrocytes.