Il 10 elisa kit

IL-10 and β-endorphin were measured both in primary cells and spinal enlargements of neuropathic rats. For primary cells, cell culture supernatants were collected after treatment with GW9508 for 2 h and then centrifuged at 5000 rpm at 4 °C for 5 min. The supernatants after being centrifuged were prepared as the protein sample for ELISA. For the spinal enlargements, they were isolated from neuropathic rats after drug administrations, homogenized at 4000 rpm for 15 s with a homogenizer (Fluko Equipment) in 10 mM Tris-HCl (1 g of tissue/5 ml), and centrifuged at 4000 rpm at 4 °C for 15 min. The total protein concentration was measured by using the standard BCA assay (Beyotime Institute of Biotechnology, Shanghai, China). The levels of IL-10 and β-endorphin were measured using commercial IL-10 ELISA kit (Invitrogen) and β-endorphin ELISA kit (Phoenix Pharmaceuticals) according to the operation manuals. The concentrations of IL-10 and β-endorphin were calculated by a calibration curve performed at the same time. The linear range was 1–500 and 1–100 pg/ml for the IL-10 and β-endorphin assay, respectively.

1 ml of 3% Thioglycollate was injected into the peritoneum of mice and animals were culled either 18 hours (neutrophils) or 4 days (macrophages) later. For neutrophil purification the peritoneum was flushed with 5 ml of PBS containing 2 mM EDTA, cells were then separated and re-suspended in complete culture medium (DMEM with 10% FBS) in a tissue culture flask for 2 hours to remove adherent cells. Non-adherent cells (neutrophils) were removed and re-suspended to 5 × 10⁶ cells/ml and aged overnight. Preparations of neutrophils were routinely of >90% purity as assessed by FACS. Macrophages were isolated with the same approach: following incubation on a tissue culture flask non-adherent cells were removed to leave adherent macrophages (100,000/well). These were then washed and incubated with aged neutrophils (200,000/well) for either 12 or 24 hours. At each stage cell samples were taken for cytospin analysis to confirm cellular purification. Culture medium was then removed and stored at −80 °C before analysis undertaken using an Il-10 ELISA kit, as directed (Affymetrix, eBioscience).