Bms630

After euthanizing, treated joints with surrounding synovial tissues were dissected and frozen in liquid nitrogen, pulverized by a mortar and pestle, and homogenized in a lysis buffer (89901, Thermo Scientific, USA) containing protease and phosphatase inhibitors (78442, Thermo Scientific, USA) and centrifuged for 10 min at 400 g (4°C) to collect the supernatants. IL‐1β concentration was quantified by ELISA (BMS630, Thermo Scientific, USA).

IL-18 and IL-1β levels from the supernatant of homogenized cultured cells were measured by ELISA. An IL-1β ELISA kit (eBioscience; Ref: BMS630, Lot: 87225015) and an IL-18 ELISA kit (Novex; Ref: KRC2341, Lot: 130401/A) were used for the analysis. Each sample was tested at least three times.

Inflammation markers (TNF-α, IL-1β,IL-18) were measured in renal homogenates and plasma samples by using commercial kits. The kits used in the measurement have the following brand, reference and lot numbers: TNF-α (eBioscience, Ref: BMS622, Lot: 91475038), IL-1β (eBioscience, Ref: BMS630, Lot: 87225015), IL-18 (Novex, Ref: KRC2341, Lot: 130401/A). Cytokine levels were given as pg/mL for plasma and pg/ mg-protein for renal homogenates.

At 24 h and 72 h post-SAH, rats were anesthetized and transcardially perfused with 250 mL PBS, the left hemisphere was harvested immediately and homogenized in 0.9% normal saline at 200 mg/mL and centrifuged at 12,000×g for 20 min. The supernatant was collected and stored in − 80 °C until further used. The concentrations of TNF-α, interleukin (IL)-6, IL-1β, and IL-10 in brain tissue lysates were analyzed using commercial ELISA kits (BMS625, BMS630, and BMS629, Invitrogen) according to the manufacturer’s instructions. The final concentration of cytokines was interpolated from the determined standard curve of absorbance.

To determine the Iba1, CD86, IL-6, and IL-1β protein concentrations within the hippocampus, an enzyme-linked immunosorbent assay (ELISA) was used. Rats were anesthetized with isoflurane using rodent anesthesia vaporizer (VetFlo, Kent Scientific Corporation, Torrington, CT, USA) and each hippocampus was quickly extracted, frozen in liquid nitrogen, and stored at a temperature of −70 °C. The following ELISA kits were used for quantification of Iba1 (NBP2-66675, NovusBio), CD86 (RAB0887-1KT, Sigma-Aldrich, St. Louis, MO, USA), IL-6 (ERA32RB, Invitrogen), and IL-1beta (BMS630, Invitrogen) according to the manufacturer’s instructions. For analysis, we used both right and left hippocampi. The homogenization buffer consisted of 100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, and 0.5% sodium deoxycholate, with a protease inhibitor cocktail (cOmplete, Sigma-Aldrich, St. Louis, MO, USA). The BCA Protein Assay Kit (Pierce, Rockford, IL, USA) was used for determining protein concentration. The optical density was measured in an iMark plate spectrophotometer (Bio-Rad, Hercules, CA, USA) at a wavelength of 450 nm.

At 24 h after SAH, rats were deeply anesthetized and serum samples from the left hemisphere were obtained and stored at −80°C until use. The concentrations of IL-10, IL-1β, and IL-18 in brain tissue lysates were analyzed using commercial ELISA kits (BMS625, BMS630, and BMS629, Invitrogen) according to the manufacturer's instructions. The final concentration of cytokines was obtained from the determined standard curve of absorbance.

At 24 h post-SAH, rats were anesthetized and transcardially perfused with 250 mL pre-cooled PBS (0.1 M, pH 7.4). Brains were then removed and rapidly divided into the left and right hemisphere; the left hemisphere was frozen in an ultra-low temperature refrigerator, homogenized in PBS (0.1 M, pH 7.4) at 200 mg/mL, and centrifuged at 12,000 g for 15 min. The liquid supernatant was collected and stored at −80 °C until further use. The concentrations of TNF-α, IL-1β, IL-17and IL-18 were analyzed using specific ELISA kits (BMS625, BMS630, and BMS629, respectively, Invitrogen) according to the manufacturer’s instructions. The concentration of cytokines was expressed by the intensity of absorbance measured by spectrometry on a microplate reader (Varioskan Lux, Thermo Scientific). Results were expressed in picograms per milliliter (pg/ml).