Poly d lysine coated 96 well plate

HEK293T cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen, 11995081) supplemented with 100 μg/mL streptomycin and 100 units/mL penicillin (Gibco, 15140122), and 10% fetal bovine serum (FBS) (Invitrogen, 10437-028). Cells were maintained under standard tissue culture conditions (5% CO₂, 37 °C). PolyJet™ (SignaGen Laboratories, SL100688) transfection reagent was used for transient cell transfection. For detection of relative cellular viability levels, cells (10000/well) were seeded into poly-d-lysine coated 96-well plates (Falcon, 353072) and treated as described. Then, MTT (5 mg/mL, 10 μL/well) (MedChemExpress, HY-15924) was added. After incubation at 37 °C for 3–4 h, DMSO was added to dissolve the precipitate, and the absorbances were determined at 570 nm by a Tecan Infinite M1000 Pro plate reader.

Mouse neuroblastoma Neuro2A cells (ATCC-CCL 131) were grown in Dulbecco’s MEM (Life Technologies) containing 10% fetal bovine serum with 100 units/mL penicillin and 100 mg/mL streptomycin (Life Technologies). The cells were maintained at 37 °C in a humidified atmosphere with 5% CO₂ and 95% air. For measurement of ROS generation, the cells were seeded at 2.0 × 10⁴ cells/well in poly-D-lysine-coated 96-well plates (BD Falcon, Franklin Lakes, NJ, USA). After plating, the cells were maintained in Dulbecco’s MEM (without phenol red or serum, Life Technologies) with 100 units/mL penicillin and 100 mg/mL streptomycin for more than 16 hours until subsequent experiments. Intracellular ROS generation caused by oxaliplatin in neuro2A cells was determined using CM-H₂DCFDA. CM-H₂DCFDA is converted by an intracellular esterase into 2’,7’-dichlorodihydrofluorescein (DCFH). The non-fluorescent DCFH is then oxidized by ROS to the highly fluorescent compound 2’,7’-dichlorofluorescein. The cells were incubated for 30 minutes with 10 μM CM-H₂DCFDA at 37 °C, followed by treatment of drugs for 1 hour. Fluorescence intensity was measured using a microplate reader (Infinite M200; Tecan Japan Co., Ltd., Kanagawa, Japan) at 485 nm (excitation) /520 nm (emission).

293A cells (Life Technologies Corp.) were cultured in D-MEM medium containing 2 mM L-glutamine and 10% FBS. Cells were seeded at a density of 1 x 10⁴ cells/well in poly-D-lysine-coated 96-well plates (BD Falcon, Franklin Lakes, NJ). After ~24 h of culture at 37°C, transient transfection with human TRPV1 or TRPV1 mutants was performed by using FuGENE HD Transfection Reagent (Promega, Madison, WI) in accordance with the manufacturer’s instructions. After transfection, cells were cultured for 40–48 h in a CO₂ incubator and used for the Ca²⁺ flux assay.