For in vitro experiments, AMD3100 (1 µM; Sigma-Aldrich), anti-HGF (20 µg/ml; R&D Systems), PF184 (500 nM; Tocris), or FR182024 (10 µM; Merck Millipore) was added to cell culture media 4 h before infection. Each inhibitor and medium were changed at 0, 4, and 24 h after infection.
For in vivo experiments, AMD3100 (10 mg/kg) was intraperitoneally injected into mice at 24 and 12 h before infection. After intravenous infection of sporozoites, mice were injected with AMD3100 at 0, 12, and 24 h after infection. According to the manufacturer’s protocol for AMD3100 (also known as Plerixafor; Genzyme Plerixafor Injection Investigator's Brochure, 2009, version 13, May 27, 2009), a half-life of AMD3100 was previously shown to be ∼5–6 h. Also, the manufacturer reported that the effect of AMD3100 in vivo continues four half-life times (∼20–24 h). In addition, our preliminary experiments to test how often normal mice can be treated with AMD3100 suggested that mice treated with AMD3100 (10 mg/kg) every 6 h become unhealthy. Therefore, we decided to administer AMD3100 treatment in mice every 12 h; treatments were performed at 24 h and 12 h before infection and at 0 h, 12 h, and 24 h after infection.
For in vivo experiments, AMD3100 (10 mg/kg) was intraperitoneally injected into mice at 24 and 12 h before infection. After intravenous infection of sporozoites, mice were injected with AMD3100 at 0, 12, and 24 h after infection. According to the manufacturer’s protocol for AMD3100 (also known as Plerixafor; Genzyme Plerixafor Injection Investigator's Brochure, 2009, version 13, May 27, 2009), a half-life of AMD3100 was previously shown to be ∼5–6 h. Also, the manufacturer reported that the effect of AMD3100 in vivo continues four half-life times (∼20–24 h). In addition, our preliminary experiments to test how often normal mice can be treated with AMD3100 suggested that mice treated with AMD3100 (10 mg/kg) every 6 h become unhealthy. Therefore, we decided to administer AMD3100 treatment in mice every 12 h; treatments were performed at 24 h and 12 h before infection and at 0 h, 12 h, and 24 h after infection.