Trypsin 1 mm edta

Furanodiene (FUR) was purchased from the National Institutes for Food and Drug Control. The Roswell Park Memorial Institute-1640 (RPMI-1640) was used as the culture medium and was purchased from Gibco (Gaithersburg, MD, United States). Fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin-streptomycin (PS), and 0.25% (w/v) trypsin/1 mM EDTA were obtained from Invitrogen (Carlsbad, CA, United States). 3-[4, 5-Dimethyl-2-thiazolyl]-2, 5-diphenyltetrazolium bromide (MTT), 2′, 7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA), CellROX^®Deep probe, propidium iodide (PI), and Annexin V/PI detection kit were obtained from Molecular Probes (Eugene, OR, United States). Doxorubicin (DOX) and tert-Butyl hydroperoxide (TBHP) solution were supplied by Sigma-Aldrich (St. Louis, MO, United States). TNF-α immunoassay kit was purchased from R&D Systems (Minneapolis, MN, United States). Radioimmunoprecipitation assay (RIPA) lysis buffer and primary antibodies against TNF-R1 and p65 were obtained from Santa Cruz (Santa Cruz, CA, United States). Primary antibodies against p-IKKα/β (Ser176/180), IKKα, IKKβ, Bcl-xL, Bax, Bad, Caspase-7, Caspase-8, PARP, GAPDH, and β-actin, as well as the secondary antibodies were purchased from Cell Signaling (Danvers, MA, United States). siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States).

Adult Lister Hooded rats were deeply anesthetized by inhalation of isofluorane and euthanized by cervical dislocation. Bone marrow was extruded from femurs and tibias and seeded into plastic culture flasks at a density of 1×10⁶ cells/cm² in DMEM-F12 containing 10% fetal bovine serum (FBS), penicillin (100U/mL) and streptomycin (100 µg/mL, all from Invitrogen Inc., Carlsbad, CA, USA) and kept in an incubator at 37°C and 5%/95% CO₂/air. After 48 h, non-adherent cells were removed by washing with phosphate-buffered saline (PBS) and the medium was completely changed every 2–3 days. Cells were grown until approximately 90% confluent and then passaged (0.25% trypsin 1 mM EDTA, Invitrogen Inc.) and plated at a density of 7×10³ cells/cm². For cryopreservation, cells were frozen at the first or second passages in FBS with 10% dimethyl sulfoxide (DMSO; Merck KGaA, Darmstadt, Germany), using a Bio-Cool Controlled Rate Freezer (SP Scientific, Warminster, PA, USA), and then stored in liquid nitrogen. For all the experiments, thawed cells were cultured and used from the third to fifth passages.

The collected synovial tissues were minced and digested with 1 mg/mL collagenase type 1 (Invitrogen, Life Technologies, Nærum, Denmark) and 1% penicillin/streptomycin in Dulbecco’s modified Eagle’s medium (DMEM, high- glucose Invitrogen, USA) for 2 h at 37°C; the cells were collected after centrifugation and cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin in 5% CO₂ at 37°C. The culture medium was aspirated after 24 h to remove non-adherent synoviocytes and was changed every 48 h until the synoviocytes reached confluence. Then, the synoviocytes were passaged using 0.25% trypsin/1 mM EDTA (Invitrogen). The cells were cryopreserved between passage 3–8, and cells from these passages were used for all experiments [21 (link)].

Mixed glial cultures containing astrocytes, pericytes, endothelial cells, and microglia were isolated from human brain tissue as described previously [47 (link)] and used at passage two. Isolated pericyte cultures were generated from these initial mixed glial cultures by subsequent passaging in order to dilute out non-proliferating microglia, astrocytes, and endothelial cells as described previously [48 (link)]. Isolated microglial cultures were generated as described previously [49 (link)]. Cells were harvested using 0.25% trypsin- 1 mM EDTA (Gibco, CA, USA) with mixed glial cultures and microglia cultures also utilising gentle detachment with a cell scraper (Falcon, MA, USA) due to strong microglial attachment. Viability was determined by trypan blue exclusion (Gibco). Mixed glial and pericyte cultures were plated at 15,000 cells/cm² and isolated microglia were plated at 30,000 cells/cm² in Nunc™ microwell plates with Nunclon™ Delta surface (Nunc, Denmark). All cultures were maintained in DMEM/F12 (Gibco), 10% fetal bovine serum (FBS; Moregate, Australia) and 1% penicillin streptomycin glutamine (PSG; Gibco).

EVO (98% purity) was purchased by Sigma-Aldrich. DOX (98% purity) was obtained from Meilun Biology Technology Company (Dalian, China). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (PS), phosphate-buffered saline (PBS), propidium iodide (PI) and 0.25% w/v trypsin/1 mM EDTA were purchased from Gibco Life Technologies (Grand Island, USA). The lactate dehydrogenase (LDH) release detection kit was obtained from Roche Diagnostics. Hoechst 33342 and 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide (MTT) were obtained by Molecular Probes (Grand Island, USA). Primary antibodies against cleaved caspase-7, cleaved caspase-9, cleaved PARP, Ras, phosphorylated MEK, MEK, phosphorylated ERK1/2, ERK1/2, XIAP, cIAP1, survivin, P-gp and GAPDH and secondary antibodies were purchased from Cell Signaling Technology.