Hanks balanced salt solution (hbss)

Fura-2-AM (Abcam) is a membrane-penetrating derivative of the radiometric Ca²⁺ indicator used to measure cytoplasmic Ca²⁺ concentrations by fluorescence. Ca²⁺ imaging of Fura-2AM-loaded NHDFs was performed using a CELENA^® S digital imaging fluorescence microscope (Logos Biosystems, Daejeon, Korea) with excitation at a wavelength of 405 nm and fluorescence emission measurements at a wavelength of 519 nm. NHDFs on the confocal dish (SPL, Pocheon, Korea) were loaded for 30 min in the dark at room temperature in Hanks’ Balanced Salt Solution (HBSS) (Welgene) containing 2 μM Fura-2AM. The dish was then rinsed thrice with HBSS. Next, the NHDFs were again incubated for 30 min at 37 °C. Finally, they were washed with prewarmed HBSS, and the live NHDFs were imaged using a fluorescence microscope.

Carnosic acid was purchased from Cayman Chemical (Ann Arbor, MI, USA). Fetal bovine serum (FBS) was obtained from Biowest (Riverside, MO, USA). Dulbecco`s modified Eagle`s medium (DMEM), bovine calf serum (BCS), Fetal bovine serum (FBS), trypsin-EDTA, phosphate-buffered saline (PBS, pH 7.4), and Hank`s balanced salt solution (HBSS) were purchased from Welgene Inc. (Gyeongsan, Korea). Dimethylsulfoxide (DMSO), antibiotic-antimycotic solution 100X, insulin, 3-isobutyl-1-methyl-xanthine (IBMX), dexamethasone, isopropanol, formaldehyde, DCFH-DA, DHE, protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), Triton X-100, 1,4-dithiothreitol (DTT), skim milk, and DPAI (4′,6-diamidino-2-phenylindole) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies of Nox4, p47^phox, p22^phox, NF-κB (p65), phospho-NF-κB (p65), IκBα, phospho-IκBα, C/EBPα, C/EBPβ, C/EBPγ, HO-1, γ-GCSc, GST, and Nrf2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-adiponectin was obtained from Cell Signaling Technology (Beverly, MA, USA). 3T3-L1 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA).

GBL powder (batch number: GB0170920) was purchased from TnV International Inc. (Chino, CA, USA), which contained 28.52% of total flavonoid content, 6.02% of total lactone content, and 6.63% of the sum of ginkgolide A to C and bilobalide according to the information that was supplied by the vendor. Q, K, ABTS, DPPH, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), and neutral red were obtained from Sigma Aldrich Co., LLC (St. Louis, MO, USA). Acetone and dimethyl sulfoxide (DMSO) were purchased from Dae Jung Chemicals (Siheung, Korea). Phosphate-buffered saline (PBS), 1 vol% penicillin-streptomycin and Hanks’ balanced salt solution (HBSS) were obtained from Welgene Inc. (Gyeongsan, Korea). Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from GE Healthcare (Chicago, IL, USA). Nerve growth factor (NGF mouse protein 7S subunit) and 1 vol% antibiotic-antimycotic (10×) solution were purchased from GIBCO/Invitrogen (Grand Island, NY, USA). All other chemicals were of analytical and reagent grade.

The cytotoxicity of the blank and Li-DNM patches was evaluated by a colorimetric cell viability assay sensitive to lactate dehydrogenase (LDH) activity, called CCK-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), of an F11 (ECACC 08062601) cell line that derived from rat embryonic dorsal root ganglion cells [22 (link)]. Briefly, F11 cells were plated in 96 well plates (8 × 10³ cells per well) and incubated for 24 h before assay. Lidocaine powder, DMN patches with or without lidocaine, and a lidocaine cartridge used in dental clinics were diluted in serum-free Dulbecco’s Modified Eagle Medium (DMEM) media with an equal concentration of lidocaine, and F11 cells were treated on each group for 2, 3, 6, 12 and 18 h. After cells were washed with Hanks balanced salt solution (HBSS; Welgene, Daegu, Korea), one-hundred microliters of CCK-8 solution was added to each well and was incubated for an additional 1 h at 37 °C. The optical density (OD) of each well at 450 nm was measured by a microplate reader (Epoch2, Biotek, Seoul, Korea). The cell viability (relative to control) is expressed as the ratio value of (OD_test−OD_blank)/(OD_control−OD_blank), where OD_test is the optical density of the cells exposed to the test solutions, OD_control is the optical density of lidocaine-untreated cells and OD_blank is the optical density of the wells without cells.

Scopolamine hydrobromide, (−)-EGCG, Hank’s balanced salts (HBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), polyethylenimine, 1,1,3,3-tetramethoxypropane, trichloroacetic acid (TCA), thiobarbituric acid (TBA), acetylthiocholine iodide (ATCI), and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich Co., LLC (St. Louis, MO, USA). Hank’s balanced salt solution (HBSS) and minimum essential medium (MEM) were purchased from Welgene, Inc. (Gyeongsan, Korea). Horse serum and penicillin–streptomycin were purchased from Biowest (Rue de la Caille, Nuaillé, France) and Gibco BRL (Grand Island, NY, USA), respectively. Lysis buffer was obtained from Noble Biosciences, Inc. (Hwaseong, Korea). Protease inhibitor was purchased from GenDEPOT (Barker, TX, USA). An EZ-SOD assay kit (DG-SOD400) was purchased from DoGenBio Co., Ltd. (Seoul, Korea). All reagents used in this experiment were of analytical grade.