Mouse tnf alpha elisa kit

On days 1, 3, and 7 after surgery, blood was obtained via retroorbital bleeding and allowed to clot at RT. The levels of inflammation factors in sera, namely, TNF-α, IL-6, and IL-10, were measured using the Mouse TNF alpha ELISA kit (Abcam, USA), Mouse IL-6 ELISA kit (Abcam, USA), and Mouse IL-10 ELISA kit (Abcam, USA) as per the manufacturer’s instructions.

To evaluate the inflammatory response in bone defect mouse models, tail venous blood of mice was extracted on the 4th day after treatment. The supernatant was collected via centrifugation (1,000 rpm) to detect IL-1β, IL-4, and IL-10 levels by ELISA. Mouse IL-1 beta ELISA Kit (ab197742), Mouse TNF alpha ELISA Kit (ab100747), and Mouse IL-10 ELISA Kit (ab255729) were purchased from Abcam. For immune function detection, the blood samples of mice were collected to separate peripheral blood mononuclear cells (PBMC) by density gradient centrifugation, and PBMC concentration was adjusted to 1 × 10⁶/mL with binding buffer solution. A PBMC suspension (100 μL) was transferred to a 5 mL PE tube, and PE Anti-CD4 antibody [EPR20122] (ab252151) and Alexa Fluor® 647 anti-CD8 alpha antibody [EPR21769] (ab237365) were used to separately label CD4 and CD8. Then, 5 μL of annexin V-FITC and 5 μL of propidium iodide were placed to the above mixture solution. After mixing and incubating in the dark for 15 min, flow cytometry (Attune CytPix, Thermo Fisher Scientific) was used to detect lymphocyte apoptosis.

Blood samples were obtained by cardiac puncture and collected in heparinized tubes [36 (link),60 (link)]. BAL samples were obtained according to the technique developed in the Laboratory Immunobiotechnology of CERELA-CONICET (San Miguel de Tucuman, Argentina) [38 (link),61 (link)]. The trachea was exposed and intubated with a catheter, and then two sequential bronchoalveolar lavages were performed on each mouse by injecting sterile PBS. The recovered fluid was centrifuged for 10 min at 900× g and the fluid frozen at −70 °C for subsequent cytokine determinations. Tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin 10 (IL-10), IL-1β, IL-6, IL-17, KC, and monocyte chemoattractant protein 1 (MCP-1) concentrations were determined in serum and BAL samples using commercial ELISA kits. IFN-γ (Mouse IFN-gamma Quantikine ELISA Kit, sensitivity: 2 pg/mL), IL-10 (Mouse IL-10 Quantikine ELISA Kit, sensitivity: 5.2 pg/mL), IL-1β (Mouse IL-1β DuoSet ELISA, sensitivity: 1.5 pg/mL), IL-6 (Mouse IL-6 Quantikine ELISA Kit, sensitivity: 1.8 pg/mL), and IL-17 (Mouse IL-17 Quantikine ELISA Kit, sensitivity: 4.7 pg/mL) from R&D Systems (USA). TNF-α (Mouse TNF alpha ELISA Kit, sensitivity: 9.1 pg/mL), MCP-1 (Mouse MCP1 ELISA Kit, sensitivity: 0.487 pg/mL), and KC (Mouse KC ELISA Kit, sensitivity: 1.95 pg/mL) kits were obtained from Abcam (Cambridge, UK).