stages, and thus with a high content of paramagnetic hemozoin, were retained in the column [29], while non-magnetic early-stage parasites passed through and were collected, washed in PBS/2% BSA, centrifuged at 500g for 5 minutes at 25uC and put back into culture with 40 ml/50 ml medium of gentamycin (Gibco, UK). These synchronsied cultures were left to grow for ,6 hours to ensure viability before gametocyte cultures were established from them. Purification of mature gametocytes Gametocytes were produced as described elsewhere [30,31] with the following modifications. A magnet-synchronised asexual culture at approximately 6% parasitaemia was used to start 15 ml cultures at a 0.6% parasitaemia and 6% haematocrit. Medium was changed every day until parasitaemia reached 15–20% at approximately day 5, and medium was added to a volume of 25 ml, and thus a haematocrit of 3.6%. N-acetyl glucosamine (NAG) was added at a concentration of 50 mM to kill asexual stages at approximately day 8 [32–34] and maintained at this concentration throughout the period of gametocyte culture. Fully mature gametocytes were harvested 17 days after initiation of the asexual culture, and purified by magnetic separation through a MACS column as described previously [35]. Gametocyte maturity was checked by successfully inducing ex-flagellation as described elsewhere [30,36]. Earlier stages of gametocyte development were synchronized and purified as previously described [37]. Flow cytometry The presence of antibodies in plasma recognizing surface antigens on parasite-infected RBCs at different stages of development was demonstrated using an adaptation of a previously established flow cytometry protocol for asexual parasites [35]. In brief, cultures were magnet-enriched and labelled with the nuclear stain ethidium bromide (EB) at 0.1mg/ ml per 105 erythrocytes, differentiating infected (nucleated) from non-infected (anucleate) erythrocytes. Aliquots (100 ml) of live, stage-enriched parasite-infected RBCs were incubated for 30 min each with test plasma (5 ml), goat anti-human plasma (diluted 1:2000) and fluorescein isothiocyanate (FITC)-conjugated rabbit anti-goat immunoglobulin (diluted 1:25). Samples were washed 3 times between incubations in phosphate buffered saline supple- mented with 2% foetal calf serum and kept overnight at 4u C before flow cytometry analysis (FACS Scan, Becton Dickinson). Before each experiment with each of the parasite stages, compensation to exclude background flourescence on the vertical and horizontal axes was carried out on samples single-labelled with EB or FITC, respectively. For gametocyte stages, plasma samples from all 200 Gambian children were tested at the same time, together with control sera. To check that erythrocytes had not ruptured and thus released free gametes, representative samples were examined after flow cytometry to confirm that erythrocyte rupture had not occurred. In addition, side-scatter measurements indicated that host cells remained intact in our flow cytometry experiments. Asexual parasites were tested in 3 separate experiments, with plasma samples batched by year of collection. Data were analyzed using WinMDI software. EB-positive parasite- infected erythrocytes were gated and used to create a 2- dimensional dot blot for each individual sample. Antibody binding was estimated in terms of the percentage cells labelled with both EB and FITC, read in the right upper quadrant (RUQ) of the dot plot. All results shown were confirmed in at least two experiments with the same reagents, but using independent gametocyte preparations. Data analysed are from one of those experiments. Gametocyte Surface Antigens We defined a simple cut-off for positivity. For mature gametocytes, all 200 plasma were run in a single experiment with negative controls in parallel. The mean proportion of events in the RUQ quadrant among the six negative control (unexposed European) plasma was 0.20, with a standard deviation of 0.0305. Plasma were defined as anti-GSA positive sera if with mature gametocytes the proportion of events in the RUQ was above a value of 0.261 (i.e. 26.1% of gametocytes being bound by IgG), representing the mean of the negative controls plus two 2 SD. For asexual parasites, plasma were tested in separate experiments for each year of collection, with negative controls run in each experiment. Mean proportion RUQ for asexual recognition was 0.134, 0.129 and 0.207 for the six negative controls in experiments testing plasma from 2000, 2001 and 2002 respectively. Positivity was then assigned as for gametocytes using the relevant mean plus two SD as cut-off, calculated separately for each of these three experiments. Immunofluorescence antibody visualisation of immature gametocytes Aliquots of early stage gametocytes from the starter flask for subsequent experiments were acetone-fixed on glass slides and stored at 220C. Parasites were counter-stained with ethidium bromide and the incubated with rabbit polyclonal sera recognising Pfs16 (kind gift of David Baker), washed, and incubated with goat anti-rabbit IgG (H _ L), conjugated to AlexaFluor 488 (Cambridge Biosciences). Immunofluorescence was then visualised by confocal microscopy as previously described [34]. ELISA for antibodies to Pfs48/45 and Pfs 230 The presence of human antibodies of the IgG1 isotype specific for Pfs230 and Pfs48/45 was determined as previously described [38]. Positive and negative controls were included on each plate. A relative recognition ratio was estimated for each plasma for each of the two sexual stage antigens Pfs48/45 & Pfs230 thus: (OD in the ELISA test well–OD in control (no primary antibodies) well)/OD in control well. This was analysed as a continuous variable. Data analysis Statistical analysis was performed using Stata (SataCorp. Stata Statistical Software: Release 8.0, Collage Station, TX: Stata Corporation). Normally distributed continuous variables were compared between categories by Student’s t-test; gametocyte densities and oocyst densities were analysed by comparing the ratio of arithmetic means in negative binomial regression as previously described [23,24]. The x2 (chi) test was used to compare proportions, except where expected values in the 262 tables were ,6, in which case Fisher’s exact test was used. Results Purity of synchronous gametocyte cultures Immunofluorescence experiments were performed to verify that asexual parasites had been eliminated from the starting gameto- cyte culture at day 8, as stage I gametocytes are difficult to discriminate from early trophozoites of the asexual lineage [39]. The majority of ethidium-bromide stained, and therefore nucleated, erythrocytes examined were found to be positively stained with anti-Pfs16 antibody (Fig. 1a). Asexual parasites were estimated to account for fewer than 1% of parasitised cells at this stage. The culture flasks established from these putative gameto- cyte cultures were examined 2 days later and found to harbour well-synchronised gametocytes at stage IIb (Fig. 1b). PLoS ONE | www.plosone.org 3 May 2008 | Volume 3 | Issue 5 | e2280