Benfotiamine

BV-2 microglial cell line was developed by immortalizing primary mouse microglial cells with v-raf/v-myc recombinant retrovirus, in the laboratory of Dr Blasi [36 (link)] and was a generous gift from Dr Alba Minelli (University of Perugia, Perugia, Italy). Cells were maintained in RPMI 1640 medium (GE Healthcare Life Sciences, Freiburg, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS, PAA Laboratories GmbH, Pasching, Austria) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified incubator under a 95% air/5% CO₂. When cells reached approximately 80% confluence, they were detached with 0.1% trypsin-EDTA (PAA Laboratories GmbH, Pasching, Austria), seeded into appropriate dishes and incubated overnight. Then BV-2 cells were pre-treated for 30 min with different concentrations of benfotiamine (Sigma-Aldrich, Munich, Germany; 50, 100 or 250 μM) before stimulation with LPS from Escherichia coli serotype 026:B6 (Sigma-Aldrich, Munich, Germany; 1μg/ml). Incubation time with LPS varied depending on the purpose of the experiment.

Assorted concentrations (1 nM–1 μM) of compounds (benfotiamine and capecitabine (Sigma-Aldrich, USA) prepared in 0.1% DMSO was used in triplicate for the primase inhibition studies, and the inhibition reaction was set up similarly to that of the primase assay. A negative control of 0.1% DMSO was introduced concerning the drug, whereas an optimized primase reaction (Methods 2.11) was taken as the positive control. The reaction mixture was maintained at 22° C for 1 h. Three volumes of the malachite green reagent (0.0812% malachite green 2.32% w/v polyvinyl alcohol, 5.72% in 6 M of HCl ammonium molybdate and water in the ratio 2:1:1:2 respectively) were added, trailed by 10% sodium citrate to the reaction to develop colour and absorbance was measured at 650 nm. GraphPad prism vs 9.0 (http://www.graphpad.com/) was utilized to calculate the IC₅₀ values of enzyme inhibition^{66 (link)}. The triplicate of the assay was used as an experimental parameter.