Mouse il 10 elisa

Briefly, the tibias and femurs of the mice were isolated, the muscle removed and the ends of the bones were cut off. The bone marrow was collected by rinsing the shaft with 2-3 ml PBS per bone using a 25 g needle. The bone marrow suspension was aliquoted and shock-frozen in liquid nitrogen. Samples were thawed, sonicated 3-times for 10 seconds each using a tip sonicator (Ultrasonic GE50) to achieve complete lysis and homogenization of the samples. The aliquots were analyzed using ELISA for cytokines (CCL5, SDF-1, IL-6, Osteopontin, IL-10, TNF-α, IL-17, IL-23), arginine, PGE₂ and arginase-1 concentrations. ELISA assays were performed exactly following the instructions of the vendors. The mouse CCL-5 ELISA kit was from Abcam (# ab100739), mouse CCR5 ELISA from Aviva Systems Biology (# OKEH 03441), mouse Arginase-1 ELISA from Abcam (# ab269541), mouse SDF-1 ELISA from Abcam (# ab100741), mouse Osteopontin ELISA from Abcam (# ab100734), mouse IL-6 ELISA kit from R&D (# M6000B), mouse IL-1 ELISA from R&D (# MLB 00C), mouse IL-10 ELISA from R&D (# M1000B), mouse IL-17 ELISA from R&D (# M1700), mouse IL-23 ELISA from R&D (# M2300), mouse TNFα ELISA from R&D (# MTA 00B) and the PGE₂ ELISA from R&D (# KGE004B). Arginine was quantified using a commercial assay from abcam (# ab241028). All values were normalized to protein in the samples.

Briefly, the tibias and femurs of the mice were isolated, the muscle removed and the ends of the bones were cut off. The bone marrow was collected by rinsing the shaft with 2-3 ml PBS per bone using a 25 g needle. The bone marrow suspension was aliquoted and shock-frozen in liquid nitrogen. Samples were thawed, sonicated 3-times for 10 seconds each using a tip sonicator (Ultrasonic GE50) to achieve complete lysis and homogenization of the samples. The aliquots were analyzed using ELISA for cytokines (CCL5, SDF-1, IL-6, Osteopontin, IL-10, TNF-α, IL-17, IL-23), arginine, PGE₂ and arginase-1 concentrations. ELISA assays were performed exactly following the instructions of the vendors. The mouse CCL-5 ELISA kit was from Abcam (# ab100739), mouse CCR5 ELISA from Aviva Systems Biology (# OKEH 03441), mouse Arginase-1 ELISA from Abcam (# ab269541), mouse SDF-1 ELISA from Abcam (# ab100741), mouse Osteopontin ELISA from Abcam (# ab100734), mouse IL-6 ELISA kit from R&D (# M6000B), mouse IL-1 ELISA from R&D (# MLB 00C), mouse IL-10 ELISA from R&D (# M1000B), mouse IL-17 ELISA from R&D (# M1700), mouse IL-23 ELISA from R&D (# M2300), mouse TNFα ELISA from R&D (# MTA 00B) and the PGE₂ ELISA from R&D (# KGE004B). Arginine was quantified using a commercial assay from abcam (# ab241028). All values were normalized to protein in the samples.

Ethanol (HPLC grade), sodium selenite (Se; Na₂SeO₃), potassium sulfate (S; K₂SO₄), sodium chloride (NaCl), potassium chloride (KCl), sodium bicarbonate (NaHCO₃), urea, calcium chloride dihydrate (CaCl₂·2H₂O₂), ammonium chloride (NH₄Cl), dibasic sodium phosphate (Na₂HPO₄), monopotassium phosphate (KH₂PO₄), and magnesium chloride (MgCl₂) were obtained from Desarrollo de Especialidades Quimicas, S.A. de C.V. (Monterrey, NL, Mexico). GIBCO (Carlsbad, CA, USA) supplied Dulbecco’s Modified Eagle Medium (DMEM), Penicillin–Streptomycin antibiotic (Pen-Strep), fetal bovine serum, phosphate-buffered saline (PBS, pH 7.4), antibiotic–antimycotic, and Hank’s buffered saline solution (HBSS). The Griess Reagent System and CellTiter 96 AQueous One Solution Cell Proliferation Assay Kits were purchased from Promega (G2930, Madison, WI, USA). Desulfoglucoraphanin was supplied by Santa Cruz Biotechnology (Dallas, TX, USA). IL-2, IL-6, IL-1β, and the TNF-α MILLIPLEX MAP Mouse Cytokine/Chemokine Panel and Human/Mouse Kit were obtained from Millipore (Billerica, MA, USA). The Total COX-2 DuoSet IC ELISA and Mouse IL-10 ELISA were purchased from R&D Systems (Minneapolis, MN, USA). The other chemicals were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA)