In vitro Effects of Saponins on Lysozyme Activity of Human Cells 347 Determination of total lysozyme activity using cell lysis Cell lysis was needed to facilitate the cell membrane breakage and the release of intracellular lysozyme activity in order to determine total lysozyme activity synthesized by cells. This procedure was performed using two freeze-thaw cycles as following: cell cultures were rapidly frozen using liquid nitrogen, then immediately thawed in 37°C water bath, and vortexed briefly to help lyse cells. This freeze-thaw cycle was repeated two times to completely extract the lysozyme from inside the cells. By lysozyme assay of the whole cell lysates we determined the total amount of lysozyme activity, including lysozyme released in the culture medium and intracellular lysozyme. Determination of cell viability In order to determine the cytotoxicity of saponins, viability of cells after exposure to each saponin for the various incubation periods was measured using CASY® cell analyzer system. Cell viability is assessed based on the integrity of plasma membrane: the living cells have intact plasma membranes whereas membranes of dead cells are broken. When a cell is exposed to a low voltage field, the electric current cannot go though the intact membrane, which is an electric insulator, if it is viable. Otherwise, as the cellular membrane is broken, electric field can go through the injured cell as there are pores on their membrane. Dilution of the cell suspension sample was carried out using freshly filtered isotonic diluting solution for cell cultures specially developed for CASY® analyser (CASYton®). Fluorescence-based lysozyme activity assay Lysozyme was quantified according to a sensitive fluorescence-based method using EnzChek® kit described previously [17]. Briefly: Using 96-well black microplate, 50μl volume of cell culture supernatant was used for each reaction. Culture medium without cells was used as a control. Starting the reaction 50μl of the DQ lysozyme substrate working suspension was added to each microplate well containing the experimental or the standard curve samples. Fluorescence intensity of each reaction was measured every 5min to follow the kinetic of the reaction at 37°C for 60min, using fluorescence microplate reader with fluorescein filter (Tecan Austria GmbH). Digestion products from the DQ lysozyme substrate have an absorption maximum at ~494nm and a fluorescence emission maximum at ~518nm. Lysozyme activity levels of the experimental samples were determined from the standard curve. Lysozyme standard curve was linear with a correlation coefficient mean of R² = 0.9962. The rate of lysis of fluorescein labeled Micrococcus lysodeikticus suspension induced by lysozyme standard at different concentrations, represented in the slop values of the kinetic curve of each concentration, was linear in the range of 4 - 63 units/ml (Data not shown). Statistical analysis Data in tables and figures were presented as mean ± SD. Differences between groups were assessed by the Mann-Whitney U test. A probability of p < 0.05 was considered significantly different. Sci Pharm. 2011; 79: 337–349