haematocrit of 5% was used for culture. Growth synchronization was achieved with 5% D-sorbitol [54]. Fluorescence Ca2+ Imaging A culture of infected erythrocytes (0.5 mL; 5 6 108 erythro- cytes/mL) was diluted 10-fold with BSA(2) medium for Ca2+ imaging (RPMI 1640 medium without phenol red; Invitrogen/ Gibco) supplemented with 25 mM HEPES, 24 mM sodium bicarbonate, 0.5 g/L L-glutamine and 50 mg/L hypoxanthine. Cultures of erythrocytes (1 mL) were collected by centrifugation (1000 g for 5 min at room temperature) and resuspended in 350 mL BSA(2) medium. Loading Solution was prepared for staining nuclei with Hoechst 33342 and loading of Fluo-4-AM [0.1 mg/mL Hoechst 33342 (Dojindo), 20–100 mM Fluo-4-AM (Invitrogen/Molecular Probes), 100-fold dilution of PowerLoad (Invitrogen) in BSA(2) medium]. A suspension of erythrocytes (350 mL) was mixed with 150 mL of Loading Solution and shaken at 200 rpm for 30–60 min at 37uC. Erythrocytes were then washed once with 10 mL BSA(2) medium (1000 g for 5 min at room temperature) and resuspended in 600 mL of BSA(+) medium [BSA(2) medium supplemented with 0.5% Albumax I and 25 mg/ mL gentamicin (Sigma)]. A suspension of erythrocytes (100 mL) was inoculated in a 35 mm glass-bottomed dish (MatTek) that had been coated with 1 mg/mL poly-L-lysine before use. After 30 min of incubation in an O2/CO2 incubator, suspended erythrocytes were removed by gentle washing with BSA(+) medium. The glass- bottomed dish was then placed in the culture chamber in which O2, CO2, temperature and humidity were maintained under conditions used for conventional in vitro parasite culture. Sequential time-lapse imaging of Hoechst 33342, Fluo-4-AM and transparent images was performed using the Leica confocal microscope system (Leica TCS SP5; Leica Microsystems) with a 636 (N.A.1.42) oil immersion objective lens and excitation at 410 nm (diode laser) for Hoechst 33342 and 488 nm (Argon laser) for Fluo-4-AM and transparent images. Emissions were collected using the true spectral detection method developed by Leica Microsystems. Images were captured every 5–15 s for 300–600 s. Specific Fluo-4 fluorescence in a parasite (F) was calculated by subtraction of background fluorescence and normalized to minimum fluorescence during the imaging period (Fmin). Inhibition of P. falciparum Development by 2-APB The 2-APB concentration used was determined by preliminary experiments with a variety of concentrations ranging from 1 to 150 mM (data not shown). Effects of 2-APB on the intraerythro- cytic parasite development were assayed using parasite cultures in the ring form stage with initial parasitaemia of approximately 1%– 2%. Cultures (500 ml) were placed in each well of a tissue culture plate (24-well flat-bottomed; Corning). 2-APB was dissolved in DMSO (Hybri-MaxH, Sigma) at 10 mM. Stock solutions were diluted with PPMI medium and added to each well of the culture plate to give a defined concentration. DMSO diluted with medium served as control. A drop of cultured erythrocytes was smeared on a glass slide and stained with Giemsa. The number of parasite- infected erythrocytes in 2000 erythrocytes was counted and defined as the level of parasitaemia. Parasite Size Calculation and Transmission Electron Microscopy Giemsa-stained smears were observed under the Nikon Eclipse 80i microscope (Nikon), photographed using the Nikon DXM 1200F camera and uploaded on a personal computer using digital photo manager software (ACT-1; Nikon). To measure parasite Plasmodium Ca2+ Oscillation in the Blood Stage size, we randomly selected 50 parasites and manually delineated areas containing parasites with lines on the screen. The area, perimeter and maximum diameter of the parasites were calculated by WinROOF software package Ver.5.8.1 (Mitani, Japan). Transmission Electron Microscopy Transmission electron microscopy was performed as previously described [55]. Specimens for transmission electron microscopy were fixed for 2 h in 2.5% (v/v) glutaraldehyde buffered with 0.1 M phosphate buffer, pH 7.4, at 4uC. They were postfixed in 1% (w/v) osmium tetroxide for 1 h. Fixed specimens were dehydrated in ascending concentrations of ethanol followed by propylene oxide for 15 min, and embedded in Epon 812. The blocks were cut with an ultramicrotome (Porter-Blim MT-2; Ivan Sorvall) with a diamond knife (Diatome). The sections were mounted on 200-mesh copper grids and stained with uranyl acetate and lead citrate, and examined under the JEOL JEM-1011 transmission electron microscope. Nucleus and ER Staining The nucleus and ER of the parasites was stained with Hoechst 33342 and ER-Tracker Red (Invitrogen). Staining with Hoechst 33342 was performed as described above for fluorescence Ca2+ imaging. ER-Tracker Red was then added at a final concentration of 0.5 mM to the erythrocyte suspension and shaken at 200 rpm for 30 min at 37uC. Erythrocytes were then washed once and resuspended in RPMI1640 medium without phenol red and observed under fluorescence microscopy (Leica DM 2500). Ethics Statement The human erythrocytes stock for the parasite culture was provided by the Hokkaido Kushiro Red Cross Blood Centre under the ethical guidelines for the blood products and obtained according to their acquisition guidelines. Written informed consent from the donor of the human erythrocytes was obtained. This study was done without a regular review from ethics committees of the Obihiro University of Agriculture and Veterinary medicine, where the parasite culture was made, because the erythrocytes stock was provided as blood product from the Red Cross after completing their ethical review for the experiment. Supporting Information Figure S1 Cytosolic calcium (Ca2+) dynamics during intraerythrocytic P. falciparum development and effects of 2-aminoethyl diphenylborinate (2-APB). (A–D) Dimethyl sulfoxide (DMSO) was used as a solvent control. Each colour represents cytosolic Ca2+ dynamics acquired from individual parasites of late ring forms (LRf), late trophozoites (LT), schizonts (S) and merozoites (M) in the presence (right columns) or absence (left columns) of 100 mM 2-APB. Embedded images in left panels are representative images of Fluo-4-loaded P. falciparum during each intraerythrocytic stage (indicated by arrowheads). Scale bars, 5 mm. (TIF) Figure S2 Subcellular Ca2+ distribution in late tropho- zoite treated with 100 mM 2-APB. The parasite Ca2+ and nucleus and were stained simultaneously with Fluo-4 (green) and Hoechst 33342 (blue). Merged images are shown in the right column (Merge). Scale bars, 5 mm. (TIF) PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e39499