Phosphate buffer saline (pbs)

PBMC samples were collected one day before surgery (day -1). Clinical data was collected on day -1 and day 8 postoperatively. We chose, based on our successful Allomap^™ biomarker test development experience[40 (link)–43 (link)], to focus on the mixed PBMC population.
Eight ml of blood was drawn into a CPT tube (Becton Dickinson, Franklin Lakes, NJ). Peripheral Blood Mononuclear cells (PBMC) from each sample were purified within 2h of phlebotomy. The collected blood was mixed and centrifuged at room temperature (22°C) for 20min at 3000RPM. Two ml of plasma was separated without disturbing the cell layer into an eppendorf tube (Sigma-Aldrich, St. Louis, MO) and stored at -80°C for future experiments. The cell layer was collected, transferred to 15ml conical tubes and re-suspended in cold Phosphate Buffer Saline (PBS) (Sigma-Aldrich, St. Louis, MO) and centrifuged for 20min at 1135RPM at 4°C. The supernatant was aspirated and discharged. The cell pellet was re-suspended in cold PBS, transferred into an eppendorf tube and centrifuged for 20min at 5.6 RPM at 4°C. The supernatant was discharged. The pellet was re-suspended in 0.5 ml RNA Protect Cell Reagent (Qiagen, Valencia, CA) and frozen at -80°C.

The MSCs harvested from sheep iliac crest were isolated according to their adherence to cell culture plastic. Bone marrow aspirates were first washed by addition of an equal volume of phosphate buffer saline (PBS; Sigma-Aldrich, France) and centrifuged at 220×g for 5 min. The cell pellets were suspended in Dulbecco’s Modified Eagle Medium (DMEM; Lonza, Germany) containing 10% heat-inactivated fetal bovine serum (Gibco, Thermo Fisher Scientific, France), 50 U/mL of penicillin (Lonza, Germany), 50 μg/mL of streptomycin (Lonza, Germany), 2.5 μg/mL Fungizone (Lonza, Germany), and seeded in a T75 culture flasks, under standard cell culture conditions. The following day, medium was discarded and attached cells were gently washed up several times with PBS to remove non-adherent cells. Flasks were then incubated for several days in DMEM, replaced every 72 h to promote emergence of colonies from adherent cells. When cells finally reached sub-confluence, they were sub-cultured until passage 2, when they were expanded for stemness characterization.

RPMI-1640 medium, penicillin/streptomycin, and fetal bovine serum (FBS) were purchased from Gibco (Invitrogen, Germany). Hooke kit for EAE induction (myelin oligodendrocyte glycoprotein_35-55 (MOG_35-55) (20 (link)) combined with complete Freund’s adjuvant (CFA) emulsion (21 (link)) and pertussis toxin (PTX):5X) and MOG_35-55 for in vitro stimulation of cells were purchased from Hooke laboratories (EK-0115, Lawrence, MA, USA). Reagents required for histopathological analyses including paraffin, xylol, alcohol, phosphate buffer saline (PBS) and Luxol fast blue were obtained from Sigma (Sigma-Aldrich, Germany). IL-6 ELISA kit and cell proliferation ELISA BrdU kit were obtained from e-Bioscience and Roche, respectively. MS14, an Iranian herbal-marine compound classified as equivalent to food with no observable adverse effect level (NOAEL), was donated by deceased Dr. Ahmadi.

Hoechst 33258 dye was from Thermo Fisher Scientific (Waltham, MA, USA). Annexin V-FITC/PI apoptosis kit was purchased from Invitrogen (Carlsbad, CA, USA). Phosphate buffer saline (PBS) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (St Louis, MO, USA). SP600125 was from Selleck Chemicals (Houston, Texas, USA). All antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Glucose, sodium hydroxide (NaOH), and phosphate buffer saline (PBS) were purchased from Sigma Aldrich (St Louis, MO, United States). Nutrient agar and bacteriological peptone, citric acid, and yeast extract were supplied by the Oxoid Ltd., while dextrose by Daejung, South Korea. Curcumin and sodium dihydrogen phosphate (NaH₂PO₄) purchased from Merck & Company, Inc. (Darmstadt, Germany).

Aluminum foils (thickness 0.5 mm, purity 99.999%) were obtained through Goodfellow Ltd. (Cambridge, UK), ethanol absolute (C₂H₅OH, 99.0%, ACS reagent), whereas acetone ((CH₃)₂CO, ACS Basic) perchloric acid (HClO₄, 70% ACS), oxalic acid (H₂C₂O₄, C.N. 144-62-7, M_w = 90.03 g/mol), (3-aminopropyl trimethoxysilane, C.N. 919-30-2, M_w = 221.37 g/mol), hydrochloric acid (HCl, 37%, C.N.7647-01-0), copper chloride (CuCl₂, C.N.-10125-13-0, >99%), Rhodamine 6G (C₂₈H₃₁N₂O₃Cl, C.N. 989-38-8, M_w = 479.01 g/mol), Polystyrene sulfonate (C₈H₈O₃S, C.N.25704-18-1, M_w ≈ 70,000), Polyallylamine hydrochloride (CH₂CH(CH₂NH₂.HCl)_n, (C.N.71550-12-4, M_w ≈ 50,000), calcium chloride (CaCl₂, C.N. 10043-52-4, ≥93% anhydrous), phosphate buffer saline (PBS, bioperformance, pH-7.4) were purchased from Sigma Aldrich (Munich, Germany). Double deionized water (DI) (18.6 MΩ, PURELAB Option Q) was used for all the solutions unless otherwise specified.