Aminoguanidine hemisulfate

No-Nitro-L-arginine methyl ester hydrochloride (L-NAME), urethane, Carbachol and aminoguanidine hemisulfate were obtained from Sigma Chem. Co. (St Louis, MO).

Mice were immunized by subcutaneous (s.c.) injection in each hind flank with an emulsion consisting of 50 μg MOG_35-55 peptide (Genscript, Piscataway, NJ) and 500 μg heat-inactivated Mycobacterium tuberculosis (Difco Laboratories, Detroit, USA) in incomplete Freund’s adjuvant (Sigma, St. Louis, MO). In addition, mice received 200 ng pertussis toxin (Sapphire Bioscience, Redfern, NSW, Australia) intraperitoneally (i.p.) on days 0 and 2 post-immunization (p.i.) as previously described^{7 (link)}. Mice were weighed and monitored for signs of disease daily. EAE disease was scored 0-5 as follows: 0 = unaffected, 0.5 = loss of tonicity in distal region of tail, 1 = half-tail paralysis; 2 = full tail paralysis; 3 = one hind limb paralysis or severe weakness in both hind limbs; 4 = full hind limb paralysis; and 5 = moribund^{8 (link),9 (link)}. MIS416 was provided by Innate Immunotherapeutics (Auckland, New Zealand) and was administered (100 μg/mouse) weekly via the tail vein. Aminoguanidine hemisulfate (Sigma) was administered to mice (100 mM in drinking water) to inhibit inducible nitric oxide synthase (iNOS) in vivo^{9 (link)}.

CD4⁺ T cells (pooled from 1–2 mice per experiment) were isolated from 2D2 mice as described above and resuspended at 2x10⁷ cells/ml in dPBS (Life Technologies). 625 nM CFSE (Molecular Probes, Life Technologies) was added and incubated at room temperature for 8 minutes in the dark before the reaction was quenched by adding an equal volume of 100% FCS. Cells were washed once with dPBS (Life Technologies) and twice with complete medium Dulbecco’s modified essential medium, 10% FCS, 100 U/ml penicillin plus 100 mg/ml streptomycin, 10 mM Hepes, 2 mM L-glutamine and 50 μM 2-mercaptoethanol (all from Life Technologies), before being used in T cell co-cultures. For some experiments, 1 mM aminoguanidine hemisulfate (Sigma) was added to the T cell co-cultures to inhibit iNOS. Cell proliferation was assessed by flow cytometry using a Canto II flow cytometer (BD Biosciences)