Mouse tnf α elisa kit

Proteins were extracted using T-PER Mammalian Protein Extraction Reagent (Pierce Biotechnology, Rockford, IL). C1q/TNF-related protein 9 (CTRP9) and TNF-α were measured using the Mouse CTRP9 ELISA kit (Aviscera Bioscience, Santa Clara, CA) and the Mouse TNFα ELISA kit (Thermo Scientific, Seattle, WA), respectively. The total protein concentrations were measured by the Pierce BCA Protein Assay kit (Thermo Scientific). Individual protein concentrations were calculated as the abundance of specific protein constituents divided by the total protein concentrations. Serum IL-6 level was determined by using a commercial ELISA kit (Thermo Scientific).

ICR mice were intraperitoneally (i.p.) administered with SM in 10% Tween-80 at the dose of 50 mg/kg and vehicle (10% Tween-80) 1 h prior to endotoxemia induction by LPS (055:B5, Sigma-Aldrich, USA) i.p. at a dose of 5 mg/kg (non-lethal endotoxemia) or 20 mg/kg (lethal endotoxemia). We used SM at a dose of 50 mg/kg, since in our previous work [73 (link)] for the evaluation of anti-inflammatory activity of SM on the models of carrageenan- and histamine-induced paw edema, we found out that the dose of 50 mg/kg is the maximum possible dose for SM that can be administered in mice without significant toxicity. Blood samples were collected from the retro-orbital sinus 4 h after LPS injection at a dose of 5 mg/kg. Blood serum was prepared by a standard protocol, and the pro-inflammatory cytokines TNF-α and IL-6 were measured by a mouse TNF-α ELISA kit and a mouse IL-6 ELISA kit (Thermo Scientific, Rockford, IL, USA) according the manufacturer’s instructions. The survival rates were evaluated after LPS injection at the dose of 20 mg/kg. The mortality of mice was assessed every 12 h for 7 days. SM- and vehicle-treated mice without LPS challenge were used as controls. In both experiments, dexamethasone at the dose of 0.5 mg/kg (non-lethal endotoxemia) and 1 mg/kg (lethal endotoxemia) was used as a reference drug with proven anti-inflammatory activity.

ICR mice were intraperitoneally (i.p.) pretreated with SM in 10% Tween-80 (50 mg/kg), vehicle (10% Tween-80), or dexamethasone (0.5 mg/kg) as a reference anti-inflammatory drug 1 h prior to peritonitis induction by 1% carrageenan i.p. Saline buffer was also administered i.p. in the normal control group instead of carrageenan injection. Four hours after peritonitis induction, mice were sacrificed by cervical dislocation, and the peritoneal cavity was washed with 2 mL of heparinized cold saline buffer to obtain peritoneal exudates. The collected samples were centrifuged (2500 rpm, 5 min, 4 °C), the cell pellets were resuspended in 50 μl of PBS, and total leukocyte counts were performed with a Neubauer chamber by optical microscopy after diluting in Turk solution (1:20). The supernatants were collected to assess the levels of pro-inflammatory cytokines TNF-α and IL-6 using a mouse TNF-α ELISA kit and a mouse IL-6 ELISA kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. To determine the differential leukocyte counts, peritoneal cells were centrifuged and placed onto slides, stained with azur-eosin by the Romanovsky–Giemsa method, and examined by optical microscopy. The results were expressed as the number of total leukocytes (×10⁶/mL) and the ratio of neutrophils, lymphocytes, and monocytes (%).

The potential immune activation of cells was estimated by the concentration of tumor necrosis factor alpha (TNF-α) in the cell culture media after cultivation of RAW 264.7 cells on the tested materials and polystyrene (PS) control dishes for 7 days. Measurements were performed using the commercially available Mouse TNF-α ELISA Kit (Thermo Scientific Inc., Rockford, IL, USA, Cat. No. 1347.4 EMTNFA) in accordance with the manufacturer's protocol. This procedure has been described in detail in [19] , and is also provided in the Supporting Information S5.