5. Mouse cell isolation In NOD/SCID mice inflammation was induced by IP injection of 1 ml 3% thioglycolate (Sigma) diluted in sterile PBS. 4–5 days after, the peritoneal cavity was washed with HBSS without Ca2+ and Mg2+. The collected cells were washed twice in RPMI supplemented with L-glutamine, Penicillin (100 U/ml), Streptomy- cin (100 mg/ml), and 10% Fetal Calf Serum (FCS) and seeded at 36105 per well in a 96-well culture plate. Single cell suspensions of splenocytes were prepared in cold RPMI 1640 medium supple- mented with 10% FCS and filtered on a 100 mm cell strainer to remove debris. Erythrocytes were lysed with ACK lysis buffer, and the splenocytes were washed 2 times with RPMI supplemented with 10% FCS and seeded at 36105 per well of a 96 well culture plate. 6. Cytokines/chemokine/chemiluminesence assay 100 ml blood samples were collected from the retro-orbital plexus with a Pasteur pipette, and sera were stored at 280uC. Conditioned media obtained after 16 h stimulation of peritoneal cells and splenocytes with lipopolysaccharide (LPS, 1 mg/ml) (Sigma) were stored at 280uC. Cytokines and chemokines (IL-6, MCP-1, IFNc, TNFa, IL-12p70 and IL-10) were quantified using the BDTM Cytometric Bead Array mouse inflammatory kit (BD biosciences) following the manufacturer’s recommendations on a FACScalibur (BD biosciences). Since production of reactive oxygen intermediates (ROI) closely mirrors the state of activation of macrophages and polymorpho- nuclear cells, luminol dependent photometric assay was used to measure ROI. Blood samples were collected from NOD/SCID mice, washed with HBSS with freshly added Ca2+ and Mg2+. Washed blood was diluted 1/10 in HBSS and 90 ml blood was added to each well in a 96 well plate (Nunc, Denmark) and incubated for 30 minutes at 37uC after adding 10 ml PMA (final concentration 1 mg/ml) to stimulate the cells. 50 ml of luminol (final concentration 200 mg/ml) solution was added immediately before measuring emissions. Improved P. falciparum Mouse Model 7. Analysis of deep-seated organs for parasite differential count NSG mice were used for the comparison of parasite differential counts in the peripheral blood, with that in deep-seated organs. Four mice were infected with UPA strain, and when a parasitemia of .10% was reached, a thin smear from peripheral blood was made before killing the mouse, and harvesting its organs. Kidney, liver, spleen, lung, and brain were removed from each mouse. Parasite content was assessed from blots made by repeatedly spotting sections from each organ. These slides were then stained with Giemsa. The last blots taken were considered to be the most representative of the parasite content in the organ’s vascular bed, and were examined at 1000x magnification to perform differential counts of each stage (.200 parasites from each organ counted). Results Development of an IV model In a previous study [6], we have shown that among NOD/ SCID mice inoculated intraperitoneally with huRBC and P. falciparum (IP-IP model), the majority do not support a stable and sustained peripheral blood parasitemia. For this reason we attempted to develop a new model in which both huRBC and P. falciparum are directly injected into the mouse blood stream (IV- IV model). With this IV protocol 100% of NOD/SCID mice proved to be parasitized by day 1 post-inoculation vs only 56% using the IP protocol (data not shown). Despite this marked improvement, among the 59 mice treated with the standard immunomodulatory protocol of innate defences (clo-lip), the parasitemia duration was short (average 5.5 days) and parasitemia was stable for $12 days in only 5 mice (Figure 1). We sought to better understand this large variation in duration of parasitemia. It was not explained by differences in chimerism. However, close analysis did show a significant difference in parasitemia duration between young mice and aged mice. Figure 1. Effects of aging and inosine on parasitemia in NOD/SCID mice. Parasitemia is shown as a percentage of total erythrocytes found in mouse peripheral blood measured on giemsa-stained thin smears. Levels are shown for old mice (.15 weeks) receiving inosine (green) or without inosine (yellow) and young mice (,15 weeks) with inosine (red) or without (blue). P. falciparum 3D7 strain was inoculated on day 0. A 400 ml pellet of huRBC and clo-lip (100 ml) were administered (IV and IP respectively) 3 times a week to all mice. For the inosine groups, inosine (250 mg/kg) was administered IP daily. Results represent the mean 6 SEM from 11 different experiments, positive result/success means parasitemia lasting .12 days a) green curve: n = 41, 15 positive, 36,6% success, b) yellow curve; n = 30, 5 positive, 16,6% success; c) red curve; n = 29, 0 positive, 0% success; d) blue curve; n = 10, 1 positive, 10% success. doi:10.1371/journal.pone.0018045.g001 PLoS ONE | www.plosone.org 3 March 2011 | Volume 6 | Issue 3 | e18045