Sh sy5y cell

Human neuroblastoma SH-SY5Y cells were obtained from ATCC (Manassas, VA) and cultured in a humidified incubator at 37°C with 5% CO₂. The SH-SY5Y cells were grown in Eagle’s minimum essential medium (EMEM) supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, l-glutamine (2 mM), non-essential amino acids (1 mM), and sodium pyruvate (1 mM).
RPMI 8226 human multiple myeloma cells (ATCC, Manassas, VA, United States) were cultured in RPMI 1640 medium (Euroclone, Pero, Italy) supplemented with 10% fetal bovine serum (FBS; GibcoR, Thermo Fisher Scientific, Lisbon, Portugal), 1% glutamine, and 1% penicillin and streptomycin (Euroclone, Pero, Italy). The cells were incubated at 37°C with 5% CO₂ in a humidified incubator. (R)-ASME was dissolved in phosphate-buffered saline (PBS) at 1 g/ml concentration and then diluted directly into the culture medium to working concentrations.

Human neuro-blastoma SH-SY5Y cells were obtained from American Type Culture Collection (ATCC CLR-2266) and were grown to a monolayer in a culture medium containing Dulbecco’s minimal essential medium (DMEM) and Ham’s F12, modified with 2 mM l-glutamine and 1.0 mM sodium pyruvate and supplemented with fetal bovine serum (FBS) to 10% and streptomycin 50 mg/mL. SH-SY5Y cells were maintained at 37 °C and 5% CO₂.
For the immunofluorescent experiments, cells were seeded into round coverslips pretreated with poly-l-lysine in a 12-well plate (~ 100.000 cells/well) and incubated at 37 °C with 5% CO₂ until cells reached a ~ 70% confluency. Cells were then incubated with ~ 1.0 × 10¹¹ dual-phage for 24 h, after which they were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. After 2 washes with 1X PBS, cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature and blocked with 2% BSA for 30 min at room temperature. After washing, round coverslips were cover-slipped with VECTAshield mounting medium with DAPI (Vector Laboratories) and visualized by fluorescence microscopy using FITC and DAPI filters.

SH-SY5Y Cells (ATCC) was were maintained in Dulbecco’s modified Eagle’s medium134 (HyClone, SH30022.01B) supplemented with 4 mM L-Glutamine (Sigma) and 10% fetal bovine serum (Gibco^®), Cell lines was cultured at 37°C and 5% CO₂. Cells were treated with 50 μM and 100 μM cholesterol for 48 h, respectively. Methods of protein extraction and immunoblotting analysis are showed in Immunoblotting analysis.

SH-SY5Y cells (ATCC) were cultured in 1:1 EMEM and Hams F12 (Sigma-Aldrich), with 10% FBS (50 ml), 1% non-essential amino acids, 1% 200 mM L-glutamine, 1% penicillin/streptomycin at 37 °C with 5% CO₂. SH-SY5Y cells were neuronally-differentiated for 6 days with treatment of 10 μM retinoic acid (RA) (Sigma-Aldrich) as previously described14 (link).

Anandamide transport inhibitor AM404 and pentacyclic triterpenoid glycyrrhetic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA), and quinoline compound VB-037 was purchased from Enamine (Kiev, Ukraine). Aqueous extract from G. inflata [27 (link)] and formulated SG-Tang [25 (link),26 (link)] was provided by Sun-Ten Pharmaceutical Company (Taipei, Taiwan). Mouse BV-2 microglial cells (kind gift from Dr. Han-Min Chen, Catholic Fu-Jen University, New Taipei City, Taiwan) were routinely maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA). Human neuroblastoma SH-SY5Y cells (ATCC CRL-2266) were maintained in DMEM/Nutrient mixture F-12 (DMEM/F-12) supplemented with 10% FBS. Cells were cultured at 37 °C with 95% relative humidity and 5% CO₂.