c. adami. The resulting parasitemia was assessed by enumerating parasites in 200–1000 erythrocytes on Giemsa-stained thin blood films prepared every other day, Regorafenib research buy beginning day 5 post-infection (PI). Groups of 3–6 sex- and age-matched mice between 6 and 16 weeks of age were used in each experiment. Data are presented as the per cent parasitemia, calculated as number of parasitized erythrocytes/total number of erythrocytes ×100. Two-colour cytofluorimetric analysis
was performed on splenocyte single-cell suspensions as described previously (19). The biotinylated antibodies were anti-CD3 and anti-NK1.1 mAbs (Boehringer Mannheim, Indianapolis, IN, USA) and anti-TCRß, anti-TCRγ and a hamster immunoglobulin G (IgG) control (BD Biosciences PharMingen, San Diego, CA, USA). Streptavidin–phycoerythrin was obtained from Southern Biotechnology Associates (Birmingham, AL, USA). Fluorescein isothiocyanate-conjugated
antibodies were as follows: anti-CD3 (Boehringer Mannheim), anti-CD4, anti-CD8, anti-CD45R (B220) and rat IgG isotype controls (BD Biosciences PharMingen). Propidium iodide was added shortly before data acquisition to allow electronic exclusion of dead cells. Vorinostat mw Data were acquired on a FACScan (Becton Dickinson, Mountain View, CA) flow cytometer and analysed with the use of CellQuest and Attractors (Becton Dickinson) programs. Sera were obtained from IL-2/15Rβ−/−, IL-2/15Rβ+/− and C57BL/6 mice following the suppression of parasitemia 34 days after inoculation with 1 × 106 parasitized erythrocytes. Plasmodium chabaudi-specific antibodies in the sera of test and control mice were measured by a modification of the enzyme-linked immunoabsorbent assay (ELISA) described previously (20). ELISA plates (Easy-Wash; Corning Costar Corporation, Cambridge, MA, USA) were coated with 2.5 μg/well of a crude preparation of P. c. adami blood-stage antigen. Following overnight incubation at 4°C, wells were washed and blocked. Starting at 1/250, serial twofold dilutions of each serum sample were prepared and added to antigen-coated wells in duplicate (50 μL/well). Following a 2-h incubation at room temperature, antigen-specific antibodies were detected with a
horseradish buy Etoposide peroxidase-conjugated rabbit antibody (Zymed Laboratories, South San Francisco, CA, USA) specific for the heavy chains of mouse IgM, IgG and IgA with ABTS [2,2′-azinobis (3-ethylbenzthiazolinesulfonic acid)] as the substrate. For each serum, A405 values between 1.0 and 0.1 were plotted and titre was calculated as the reciprocal of the dilution of serum yielding an A405 = 0.2. Statistical analysis was performed with the unpaired, two-tailed, Student’s t-test and GraphPad Prism 3 software (GraphPad Software Inc., San Diego, CA, USA). A P value of < 0.05 was considered statistically significant. To determine whether the IL-2R is essential for the development of immunity against acute blood-stage infection, the time course of P. c.