The plates were rapidly shaken on a microplate shaker for 20 min to extract the NR. The absorption was measured at 545 nm in a microtiter
plate reader (spectrophotometer). check details The optical density (OD) was calculated as the difference between the absorbances at the test wavelength and that at the reference wavelength. For each concentration tested, the wells containing no cells served as reference blanks. The blood samples, obtained from three donors of two blood banks, were diluted in PBS and centrifuged at 150g for 10 min at 4 °C. The plasma and white cells were carefully removed after each wash (three times). To induce hemolysis, aliquots of terpenes diluted in ethanol (300 mM) were added to tubes containing erythrocytes suspended in PBS at a hematocrit
concentration of 50% (final volume of 100 μL). After gentle shaking, the tubes were incubated at 37 °C for 1.5 h. Subsequently, the erythrocytes were precipitated by centrifugation at 300g and 25 °C for 10 min. The magnitude of hemolysis was selleck chemicals determined spectrophotometrically at 540 nm according to the equation: %hemolysis=Aa-Ac1Ac2-Ac1where Ac1 is the control sample (0% terpene), Ac2 is the completely hemolyzed sample in Milli-Q water and Aa is the sample containing the desired terpene concentration. Terpene concentration that causes 50% hemolysis was determined in units of mM. It is well known that an average human erythrocyte occupies a volume of approximately 90 fL. The number of cells in the sample and the ratio of terpenes/cell for 50% hemolysis were calculated based on this volume. The terpenes were dissolved in ethanol to the desired concentration, and 4 μL of the solution was applied directly to the cell suspension (45 μL). The terpene-erythrocyte or terpene-fibroblast suspensions were incubated at 37 °C for 1.5 h. Subsequently, a small aliquot (∼1 μL) of the spin label 5-DSA (Fig. 1) dissolved in ethanol (5 mg/mL) was added to the cells. Each sample consisting of 5.0 × 108 RBCs or 1.3 × 107 fibroblasts in PBS containing 10% ethanol and the desired terpene concentration was introduced in capillary tube and flame-sealed for the EPR
measurement. Control samples, with Dipeptidyl peptidase and without ethanol, were measured and it was found that this concentration of ethanol did not significantly alter the membrane fluidity in either RBC or fibroblast cells. In calculating the ratio of terpene molecules/cell for each sample, the erythrocyte volume was considered to be 90 fL and for fibroblast samples the number of cells was counted. The EPR spectra were recorded using a Bruker ESP 300 spectrometer (Rheinstetten, Germany) equipped with an ER 4102 ST resonator. The instrument was programmed with the following settings: microwave power, 2 mW; modulation frequency, 100 kHz; modulation amplitude, 1.0 G; magnetic field scan, 100 G; sweep time, 168 s; and detector time constant, 41 ms. All measurements were performed at room temperature (24–26 °C).