Il 17 antibodies

For Th17 cells analysis, mononuclear cells were isolated from the brain and peripheral blood of the rat after 72 h following CA/CPR. Subsequently, 1 ml cell suspension, 50 ng phorbol 12-myristate 13-acetate (PMA, Thermo, USA), 1 μg ionomycin, and 0.7 μl monensin were added into each well of a 6-well plate and then cultured at 37 °C for 6 h. After 5 min of centrifugation at room temperature and 200×g, the cells were transferred to a flow detection tube and supplemented with a fixed membrane-penetrating agent. After washing and resuspending, the cells were incubated with antibody CD4 (1:1,000, ebioscience, USA) and IL-17 antibodies (1:1,000, ebioscience, USA), with IgG acting as the isotype control. For neuron apoptosis analysis, cells were determined by Annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Kit (ebioscience, USA). According to the manufacturer's protocol, neurons were resuspended in a binding buffer following washed once with ice-cold phosphate-buffered saline (PBS), 10 μL Annexin V/FITC was added for 10 min incubation in the dark at room temperature. Then 5 μL PI was added for 5 min. Flow cytometry (BD FACSAria2, USA) analyzed the apoptotic rate of cells.

Splenic lymphocytes were collected for flow cytometry. Prepared lymphocytes were incubated for 30 mins at 4°C in the dark for surface staining, with FITC-labeled anti-CD4 antibodies (eBioscience), PerCP-Cy5-5-labeled anti-LAP antibodies (eBioscience) or antigen presenting cell(APC)-labeled anti-GARP antibodies (eBioscience). For intracellular staining of Foxp3, lymphocytes were fixed with 4% paraformaldehyde after surface staining for CD4 and GARP and then permeabilized. Permeabilized lymphocytes were incubated with PE-labeled anti-Foxp3 antibodies (eBioscience) for 1 hours at 4°C in the dark.
For the detection of Th1, Th2 and Th17, splenic lymphocytes were stimulated by PMA (100ng/mL)/lonomycin (750ng/mL) for 4 hours in vitro. After stained with FITC-CD4, these lymphocytes were fixed and then permeabilized. The permeabilized lymphocytes were incubated with PE-labeled anti-INF-γ, IL-4 or IL-17 antibodies (eBioscience) for 30 mins at 4°C in the dark. Then, cells were washed 2~3 times and analyzed by a BD FACScan. All the results were analyzed by Flowjo 7.6.3.