Rpmi 1640 medium

BV-2 cell line was derived from primary microglial cells of C57BL/6 newborn mice infected with v-raf/v-myc retrovirus (Blasi et al., 1990 (link)). These cells express functional NADPH oxidase (Henn et al., 2009 (link)). They were a kind gift from Dr. Alba Minelli from the University of Perugia, Italy. BV-2 microglia was cultured in RPMI 1640 medium (GE Healthcare Life Sciences, Freiburg, Germany) containing 10% heat-inactivated fetal bovine serum (FBS, PAA Laboratories GmbH, Pasching, Austria) and 1% penicillin/streptomycin (Invitrogen Co, Carlsbad, CA, USA) at 37°C in a humidified incubator with 5% CO₂. Upon confluence, cells were collected with 0.1% trypsin-EDTA (PAA Laboratories GmbH, Pasching, Austria), centrifuged (500 × g for 5 min) and seeded in culture dishes, depending on the experiment. Cell were exposed to benfotiamine (50, 100, 250 μM; Sigma-Aldrich Labware, Munich, Germany) 30 min prior to stimulation with 1 μg/ml LPS (Escherichia coli serotype 026:B6; Sigma-Aldrich Labware, Munich, Germany) for 24 h or as indicated. Treatment with inhibitor of inducible NO synthase (iNOS), Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) was performed 1 h before BV-2 cells were incubated with LPS for 24 h.

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.

Mouse mammary gland adenocarcinoma tumor cell line 4T1-luc2 was obtained from Caliper(Mountain View, CA). Cells were maintained in RPMI 1640 medium(GE Healthcare) containing 10% fetal bovine serum(Sigma-Aldrich) and 2 mM L-glutamine(GE healthcare). Cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂-95% air. 4T1-luc2 was verified to be free of mycoplasma contamination.

KMRC2 (Japanese Collection of Research Bioresources Cell Bank, JCRB1011) and 786O (American Type Culture Collection, CRL-1932) were cultured in Dulbecco’s Modified Eagle's Medium (GE Healthcare, SH30243.FS) or RPMI 1640 medium (GE Healthcare, SH30027.01), respectively, supplemented with 10% fetal bovine serum (GE Healthcare, SH30396.03) and 1X Penicillin–Streptomycin (Caisson Labs, PSL01) at 37 °C in a humidified incubator with 5% CO₂.

OSCC cells SCC-25 (ATCC® CRL-1628™) and human oral keratinocytes (HOK) (ATCC® PCS-200-014™) were purchased from American type culture collection (ATCC). All cells were cultured in 5 ml plastic flasks at a cell concentration of 4 × 10⁴ in 5 ml RPMI-1640 medium (GE Healthcare Life Sciences, Little Chalfont, U.K.) containing 10% fetal bovine serum (FBS). The cells were cultured in a humidified atmosphere at 37°C containing 5% CO₂. Culture medium was changed every 2 days.