**4. Development of a measure of intraerythrocytic growth of** *P. falciparum*  **using flow cytometry and SYBR Green I**

Growth-promoting and antimalarial effects on plasmodia can be assessed both quantitatively and qualitatively by directly examining RBC smears from blood or cultures under a microscope; however, this method is tedious and subjective. Numerous novel *in vitro* assays have been introduced that are more objective, faster, more sensitive, and designed to be easier to handle. The most common of these include isotopic, enzymatic, and enzyme-linked immunosorbent assays (ELISA) (Noedl et al., 2003). Isotopic assays rely on the incorporation of radioactive 3H-hypoxanthine into the parasite DNA (Noedl et al., 2003; Webster et al., 1985; Yayon et al., 1983). These methods are relatively reliable and objective, but not sufficiently sensitive, and require the use of hazardous radioactive material. The assays are well suited for screening large numbers of compounds. Parasite lactate dehydrogenase levels have also been used to assess the growth of malarial parasites (Asahi, et al., 2005; Makler and Hinrichs, 1993; Noedl et al., 2003). ELISA-based assays can provide measures of parasite growth by quantifying biomolecules produced during parasite development, such as histidine-rich protein 2 or parasite lactate dehydrogenase, by double-

**I**ntraerythrocytic *Plasmodium falciparum* Growth in

Serum-Free Medium with an Emphasis on Growth-Promoting Factors 81

Fig. 6. Effects of various proteins on the ability of a mixture of NEFA and phospholipids to sustain growth of *P. falciparum.* Growth rate was estimated by dividing the parasitemia of

**Ring form Trophozoite Young schizont Schizont Merozoites**

Fig. 7. Different stages of the parasite cultured in the formulated CDM, stained with Giemsa.

**4.1 Optimization of flow cytometric measurement of infected RBC with SYBR Green I**  The cytometer was equipped with a single argon-ion laser tuned to a fluorescence excitation of 488 nm for 15 mW output (PAS flow cytometer, Partec Co. Ltd., Germany). A FACSCalibur (Becton Dickinson Immunocytometry Systems, USA) was also used with a single fluorescence measurement (530 nm). Analysis was performed using FCS express

the test sample 4 days after inoculation by the initial parasitemia.

software (De Novo Software Inc., Canada).

site sandwich ELISA (Druihe, et al., 2001; Noedl et al., 2002; Noedl et al., 2003). ELISA-based tests are rapid and easy to perform, and are also well suited for the screening of large number of drugs. These methods have been widely employed to detect and analyze the growth of parasites, although they are poorly suited for discriminating the developmental stages of the parasite in parasitized RBC. Flow cytometry using nucleic acid staining offers the possibility of studying the cell cycle and developmental stages of intraerythrocytic growth of malaria parasites. Flow cytometric analysis using intercalating dyes, such as acridine orange, thiazole orange, hydroethidine, propidium iodine, YOYO-1, and SYTO-16, has already been used successfully to test human and murine samples (Barkan et al., 2000; Janse and Van Vianen, 1994; Jimenez-Diaz et al., 2009; Jouin et al., 1995; Li et al., 2007; Nyakeriga, 2004; Persson et al., 2006). However, the use of flow cytometry has been limited by its lack of specificity and the complicated preparation required. We modified the flow cytometry system and introduced SYBR Green I as an intercalating dye, allowing the growth and development of *P. falciparum* to be analyzed with a high degree of accuracy (Izumiyama et al., 2009).

Fig. 5. Effects of various types of PC containing different fatty acid moieties on the abilities of NEFA to sustain growth of *P. falciparum*. Growth rate was estimated 4 days after inoculation. #, A paired NEFA of C18:1-*cis*-9 and C16:0 served as a control.

site sandwich ELISA (Druihe, et al., 2001; Noedl et al., 2002; Noedl et al., 2003). ELISA-based tests are rapid and easy to perform, and are also well suited for the screening of large number of drugs. These methods have been widely employed to detect and analyze the growth of parasites, although they are poorly suited for discriminating the developmental stages of the parasite in parasitized RBC. Flow cytometry using nucleic acid staining offers the possibility of studying the cell cycle and developmental stages of intraerythrocytic growth of malaria parasites. Flow cytometric analysis using intercalating dyes, such as acridine orange, thiazole orange, hydroethidine, propidium iodine, YOYO-1, and SYTO-16, has already been used successfully to test human and murine samples (Barkan et al., 2000; Janse and Van Vianen, 1994; Jimenez-Diaz et al., 2009; Jouin et al., 1995; Li et al., 2007; Nyakeriga, 2004; Persson et al., 2006). However, the use of flow cytometry has been limited by its lack of specificity and the complicated preparation required. We modified the flow cytometry system and introduced SYBR Green I as an intercalating dye, allowing the growth and development of *P. falciparum* to be analyzed with a high degree of accuracy (Izumiyama

Fig. 5. Effects of various types of PC containing different fatty acid moieties on the abilities

of NEFA to sustain growth of *P. falciparum*. Growth rate was estimated 4 days after

inoculation. #, A paired NEFA of C18:1-*cis*-9 and C16:0 served as a control.

et al., 2009).

Fig. 6. Effects of various proteins on the ability of a mixture of NEFA and phospholipids to sustain growth of *P. falciparum.* Growth rate was estimated by dividing the parasitemia of the test sample 4 days after inoculation by the initial parasitemia.

Fig. 7. Different stages of the parasite cultured in the formulated CDM, stained with Giemsa.
