Sc 37049

The mouse hippocampal HT22 and murine BV2 microglial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with FBS (Fetal bovine serum) (10%) and penicillin/streptomycin (1%) in a 5% CO2 incubator at 37 °C. After attaining a confluency of 70%, the cells were pretreated for 1 h with ethanol (100 µM), followed by curcumin (2 μM) or Nrf2 siRNA for 24 h, or TAK242 (TLR4 specific inhibitor). The Nrf2 gene was knocked down with Nrf2 siRNA at a concentration of 10 μM per transfection for 36 h, as directed (SC: 37049, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The transfection was conducted with lipofectamine™2000 reagent (Invitrogen, Waltham, MA, USA) when the cells culture reached to 75–80%. The control group cells were treated with 0.01% Dimethyl sulfoxide (DMSO).

HT-22 neuronal cells were cultured in 75 cm² flasks (Nunc™ EasYFlask™, Thermo Fisher Scientific, Nunc a/S, Rosklide, Denmark). Cells were counted by using a disposable hemocytometer followed by the addition of 10 μL of medium containing cells on both sides of the hemocytometer chamber. The middle central square and four 1/25 mm² corners were counted by using an Olympus microscope under 10× magnification. The cells (2 × 10⁴/mL) were further subcultured in 35-mm Petri dishes (Thermo Fisher Scientific, Nunc a/S, Rosklide, Denmark) in DMEM supplemented with 10% FBS and 1% antibiotic at 37 °C in humidified air containing 5% CO₂ according to the manufacturer’s protocol (SC: 37049, Santa Cruz Biotechnology, Inc. Dallas, TX, USA) for 36 h. The Nrf-2 was knocked down via Nrf-2 siRNA at a concentration of 10 μM/transfection. Negative siRNA (Ambion, Austin, TX, USA) was used as a control and lipofectamine™ 2000 reagent (Invitrogen) was used for transfection when the culture was 75–80% confluent. After 36 h, cells were exposed to Cd (1 μg/mL) and/or caffeine (100 μM), and in the control group to 0.01% DMSO for 24 h. After that, cells were collected in 1% PBS solution and centrifuged, followed by the removal of supernatant. Proteins were isolated by dissolving the pellets in protein extraction solution, then vertexed and ultrasonicated, and processed for Western blot analysis.