Cilostazol

Washed erythrocytes from healthy humans were diluted to a 20% hematocrit in the physiological buffer described above. Erythrocytes were incubated with either blank liposomes or liposomes containing cilostazol (Sigma, St. Louis, MO) for 30 min prior to the addition of UT-15C (100 nM, United Therapeutics, Research Triangle Park, NJ). ATP was measured prior to and at 5, 10, and 15 min after addition of UT-15C.

The PDE-3 inhibitor substance V (provided by G.J. Kumar and Dr Vaidya; 0.5 mg/kg body weight, dissolved in 0.5 % dimethyl sulfoxide [DMSO]), cilostazol (Sigma Aldrich, Germany; 10 mg/kg body weight, dissolved in 0.5 % DMSO), or vehicle (0.5 % DMSO) were given by an intraperitoneal injection before tMCAO or directly after reperfusion, that is, 60 minutes after inserting the occluding thread in the MCA.

Cilostazol was purchased from Sigma, and dissolve in DMSO at the concentration of 8×10⁴ µmol/L for storage. PBMCs or purified pDCs, cultured in RPMI 1640 (Hyclone, Logan, UT, USA) containing 10% FCS at 2×10⁵ cells or 1×10⁴ per 200 µL in round-bottomed 96-well culture plates, were pretreated with Cilostazolor DMSO as vehicle for 2 h before stimulated with 2 µmol/L CpG-A(Invivogen, San Diego, CA, USA), 2 µg/mL R848 or 5 MOI HSV. After 20h of culture, cells and supernatants were harvested for further analysis. The level of IFN-α and TNF-α in supernatant was assessed by ELISA (Mabtech, Nacka Strand, Sweden) or Flow Cytomix (eBioscience, San Diego, CA, USA). Cell viability was determined by Guava Viacount (Guava easyCyte, EMD Millipore, Billerica, MA, USA) and Trypan Blue staining.