Caspase-3 activity was determined by measuring proteolytic cleava

Caspase-3 activity was determined by measuring proteolytic cleavage of the fluorogenic caspase-3 substrate Ac-Asp-Glu-Val-Asp-AMC (Calbiochem, Laeufelfingen, Switzerland). Cells were incubated for 1 h at 37°C with 2·5 µM substrate. The cleaved reporter group fluorescence was measured at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. To quantify possible ROS generation by fibroblasts, experiments were performed measuring the oxidation of non-fluorescent 2,7′-dichlorofluorescin (DCFH) (Sigma, Buchs, Switzerland) substrate to highly fluorescent DCF by ROS. As the experimental setup could be performed only for a short exposure, LA were incubated

for 5 h with fibroblasts. Cells were loaded with DCFH during a 60-min incubation in Hanks’ balanced salt solution (HBSS; Sigma, Buchs, Switzerland) supplemented with 30 mM glucose (D-(+)-glucose (Sigma), pH 7·4, and 50 µM DCFH-DA (Sigma) at room temperature in the dark. Cells were washed three times with HBSS to remove any extracellular probe from the extracellular environment. Thereafter, cells were

exposed to various concentrations of local anaesthetic in HBSS. The amount of generated DCF was measured using a fluorescence Synergy HT (Bio-TEK, Winooski, VT, USA). The excitation filter was set at 485 nm and the emission filter was set mTOR inhibitor at 530 nm. At the same time, cell viability and activity of caspase-3 were determined. Values were expressed as mean ± standard deviation (s.d.). Results are presented as a percentage of control. Cell count and ELISA data regarding viability, proliferation rate and caspase-3 activity were analysed using three-way analysis of variance (anova). Pearson’s product–moment correlation coefficients were computed between ELISA results regarding production of ROS and cell viability. OriginPro 8G (OriginLab, Northampton, MA,

USA) and spss (SPSS, Inc., Chicago, IL, USA) were used for statistical analyses. A probability of P < 0·05 was considered statistically significant. In group 1, no negative effect of lidocaine and ropivacaine regarding cell survival was observed for the 0·3 mg/ml concentration (Fig. 1a). In the GNE-0877 presence of bupivacaine, cell death ranged between 20% and 40%. With the 0·6 mg/ml concentration, cell survival in the lidocaine and ropivacaine group was similar with 50–90%, while a prominent effect on cell death rate was observed for bupivacaine, with 30% survival after 3 days, 5% after 6 days and no survival after 9 days of incubation (Fig. 1b). In group 2, with a permanent incubation of fibroblasts with LA at a concentration of 0·3 mg/ml, 20–30% dead cells were found with lidocaine and ropivacaine after an incubation between 3 and 9 days. Cell death was more evident in the bupivacaine group, showing a time-dependent decrease of survival (Fig. 1c).

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