Odn2088

For ODN2088 (Invitrogen), in vivo ODN2088 (50 μg/mouse) was administrated via tail vein prior to instillation of mtDNA, and in vitro ODN2088 (25 μg) was added in the medium prior to mtDNA treatment. For CA074 Me (Millipore), in vitro MH-S cells were pre-treated with CA074 Me (100 μM, 40 μg) for 30 min before stimulation and then treated with CBNPs (100 µg/cm²) for 30 min with the presence of CA074 Me, and in vivo CA074 Me (40 μg/mouse) was administrated via tail vein 2 h prior to instillation of CBNPs. For SB 203580 (Sigma), in vivo SB 203580 (3.77 μg/mouse) was administrated via tail vein prior to instillation of CBNPs.

Eighteen months old C57BL/6 mice were treated with Dasatinib (D; 5 mg/kg) and Quercetin (Q; 50 mg/kg) (both from Sigma) p.o. for 3 successive days. After 4 days, mice were euthanized, and organs procured for IHC. The same treatment kinetics were applied to animals that underwent IRI to assess systemic cf-mt-DNA levels and inflammation. To monitor cardiac allograft survival, 18 months old C57BL/6 donor mice were treated with a single dose of D (5 mg/kg) and Q (50 mg/kg). Moreover, to model the clinical setting, recipient animals were subjected to weekly i.p. injections with 10 mg/kg CTLA4-Ig (Orencia) in an additional set of experiments. By 24 h, hearts were procured and transplanted. To assess the impact of cf-mt-DNA on alloimmunity, 3 months old DBA2/J recipient mice were treated daily with 30 µg of ODN2088 (Invitrogen, Carlsbad, CA, USA).