Quantifying Liver Infection Dynamics

Mouse hepatic infection was assessed at either 30 min, 2 h, 6 h, 12 h, 24 h or 46 h after P. berghei sporozoite inoculation and quantified either by real-time in vivo imaging employing the IVIS Lumina Imaging System (Caliper LifeSciences, Waltham, MA, USA), as previously described [75 (link)], or by quantitative real-time reverse transcriptase-PCR (qRT-PCR), also as previously described [79 (link)]. P. berghei bioluminescence was measured as total flux (photons/s) and analyzed with the Living Image software (version 3.0, PerkinElmer, Waltham, MA, USA). For qRT-PCR analyses, 0.7–0.9 mg of livers collected by dissection of infected mice were mechanically homogenized in TRIzol (BioRad, Hercules, CA, USA), RNA was extracted following the manufacturer’s instructions and converted into complementary DNA (cDNA) as described below. Liver P. berghei load was quantified by qRT-PCR, as previously described [80 (link)], using primers specific for P. berghei 18S rNA (Table 1). Expression of the endogenous mouse housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (Hprt) was used for normalization.