**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 software (De Novo Software Inc., Canada).

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

intensity) (Figures 9a, b).

by flow cytometry (Figures 10a,b).

Percoll sedimentation*.*

Serum-Free Medium with an Emphasis on Growth-Promoting Factors 83

Infected RBC were stained with SYBR Green I-basic by adding 8 x 105 cells into 1 ml staining solution, followed by analysis with flow cytometry. SYBR Green I-basic provided brilliant resolution of infected versus uninfected RBC and permitted visualization of infected RBC populations with high accuracy. Infected RBC were located as three clusters in twoparameter dot-plot presentations of infected/uninfected RBC from *P. falciparum* cultures: (1) cluster 1 (C1) contained predominantly ring forms with low DNA content (low fluorescence intensity); (2) cluster 2 (C2) contained predominantly late trophozoites and young schizonts with moderate DNA content (moderate fluorescence intensity); and (3) cluster 3 (C3) contained late schizonts and segmenters with high DNA content (high fluorescence

In regular cultures, schizonts burst spontaneously to release free merozoites, which then enter new RBC and increase the number of infected RBC. While the majority of released free merozoites remain in culture for a while, the merozoites after culture indicate the completion of schizogony. Parasites were synchronized at the ring form stage and cultured over 45 h under the pressure of various concentrations of the anti-schizogony drug chloroquine, to confirm the accuracy of counting the number of merozoites by flow cytometry. Merozoites released from mature schizonts were counted clearly and sensitively

Fig. 9. Two-parameter dot-plot representation of fluorescent units and side scatter from asynchronous culture (a) of *P. falciparum* and schizont-rich populations (b) separated by

In order to distinguish infected RBC or merozoites from platelets, leukocytes, and RBC debris by flow cytometry, it is essential for them to retain their normal morphological characteristics and membrane integrity, without disruption. We evaluated the effects of (1) fixatives, (2) dilution buffer and concentration range of SYBR Green I, and (3) staining period on the intensity of fluorescence of infected RBC. (1) Infected RBC in culture were fixed using fixatives such as 1% paraformaldehyde or 1% glutaraldehyde in Tris-saline solution (20 mM Tris (hydroxymethyl) aminomethane hydrochloride at pH 7.2 and 138 mM NaCl), phosphate-buffered saline (PBS) (10 mM phosphate buffer at pH 7.2 and 138 mM NaCl), or Alsever's solution. Paraformaldehyde combined with Alsever's solution proved the most useful fixative, with no noticeable lysis or deformity of infected/uninfected RBC, such as often occurs when Tris-saline or PBS is used to dilute fixatives. (2) Concentration ranges and dilution buffers for SYBR Green I were tested to determine the optimal solution for producing clearly resolved peaks of fluorescence. Individual peaks in histograms corresponding to the development stages of infected RBC showed most clearly with SYBR Green I diluted at concentrations ranging from 0.00625–2× in Tris-saline at pH 8.8 (SYBR Green I-basic). Both fixed and unfixed infected RBC gave the best results at 1× dilution of SYBR Green I. (3) The dependence of fluorescence with time for fixed or unfixed infected RBC in SYBR Green I-basic at 1× dilution was evaluated over 30 min by time-curve analysis of *P. falciparum* cultures. The frequency distribution of fluorescence was similar after staining for 5 min or longer with SYBR Green I-basic, although fluorescence could be detected within seconds after addition of the fluorescent stain to fixed or unfixed infected RBC. SYBR Green I in Tris-saline at pH 6.8 and PBS resulted in inadequate signals for infected RBC stained for less than 30 min, and in deformation and hemolysis of infected RBC. Accordingly, the optimized protocol was fixation of infected/uninfected RBC by the addition of 1% paraformaldehyde combined with Alsever's solution, and staining the fixed RBC, at measure by flow cytometry, by adding into SYBR Green I-basic solution containing SYBR Green I at x1 dilution for 5 min.

#### **4.2 Visualization of infected RBC populations with SYBR Green I-basic**

Infected RBC populations were stained as described above. All developmental stages were clearly stained with SYBR Green I with no autofluorescence of RBC (Figure 8).

Fig. 8. Developmental stages of *P. falciparum* stained with SYBR Green I-basic.

In order to distinguish infected RBC or merozoites from platelets, leukocytes, and RBC debris by flow cytometry, it is essential for them to retain their normal morphological characteristics and membrane integrity, without disruption. We evaluated the effects of (1) fixatives, (2) dilution buffer and concentration range of SYBR Green I, and (3) staining period on the intensity of fluorescence of infected RBC. (1) Infected RBC in culture were fixed using fixatives such as 1% paraformaldehyde or 1% glutaraldehyde in Tris-saline solution (20 mM Tris (hydroxymethyl) aminomethane hydrochloride at pH 7.2 and 138 mM NaCl), phosphate-buffered saline (PBS) (10 mM phosphate buffer at pH 7.2 and 138 mM NaCl), or Alsever's solution. Paraformaldehyde combined with Alsever's solution proved the most useful fixative, with no noticeable lysis or deformity of infected/uninfected RBC, such as often occurs when Tris-saline or PBS is used to dilute fixatives. (2) Concentration ranges and dilution buffers for SYBR Green I were tested to determine the optimal solution for producing clearly resolved peaks of fluorescence. Individual peaks in histograms corresponding to the development stages of infected RBC showed most clearly with SYBR Green I diluted at concentrations ranging from 0.00625–2× in Tris-saline at pH 8.8 (SYBR Green I-basic). Both fixed and unfixed infected RBC gave the best results at 1× dilution of SYBR Green I. (3) The dependence of fluorescence with time for fixed or unfixed infected RBC in SYBR Green I-basic at 1× dilution was evaluated over 30 min by time-curve analysis of *P. falciparum* cultures. The frequency distribution of fluorescence was similar after staining for 5 min or longer with SYBR Green I-basic, although fluorescence could be detected within seconds after addition of the fluorescent stain to fixed or unfixed infected RBC. SYBR Green I in Tris-saline at pH 6.8 and PBS resulted in inadequate signals for infected RBC stained for less than 30 min, and in deformation and hemolysis of infected RBC. Accordingly, the optimized protocol was fixation of infected/uninfected RBC by the addition of 1% paraformaldehyde combined with Alsever's solution, and staining the fixed RBC, at measure by flow cytometry, by adding into SYBR Green I-basic solution containing

SYBR Green I at x1 dilution for 5 min.

**4.2 Visualization of infected RBC populations with SYBR Green I-basic** 

clearly stained with SYBR Green I with no autofluorescence of RBC (Figure 8).

Fig. 8. Developmental stages of *P. falciparum* stained with SYBR Green I-basic.

Infected RBC populations were stained as described above. All developmental stages were

**ring form trophozoite schizont segmenter merozoite**

Infected RBC were stained with SYBR Green I-basic by adding 8 x 105 cells into 1 ml staining solution, followed by analysis with flow cytometry. SYBR Green I-basic provided brilliant resolution of infected versus uninfected RBC and permitted visualization of infected RBC populations with high accuracy. Infected RBC were located as three clusters in twoparameter dot-plot presentations of infected/uninfected RBC from *P. falciparum* cultures: (1) cluster 1 (C1) contained predominantly ring forms with low DNA content (low fluorescence intensity); (2) cluster 2 (C2) contained predominantly late trophozoites and young schizonts with moderate DNA content (moderate fluorescence intensity); and (3) cluster 3 (C3) contained late schizonts and segmenters with high DNA content (high fluorescence intensity) (Figures 9a, b).

In regular cultures, schizonts burst spontaneously to release free merozoites, which then enter new RBC and increase the number of infected RBC. While the majority of released free merozoites remain in culture for a while, the merozoites after culture indicate the completion of schizogony. Parasites were synchronized at the ring form stage and cultured over 45 h under the pressure of various concentrations of the anti-schizogony drug chloroquine, to confirm the accuracy of counting the number of merozoites by flow cytometry. Merozoites released from mature schizonts were counted clearly and sensitively by flow cytometry (Figures 10a,b).

Fig. 9. Two-parameter dot-plot representation of fluorescent units and side scatter from asynchronous culture (a) of *P. falciparum* and schizont-rich populations (b) separated by Percoll sedimentation*.*

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

merozoites (Figure 11).

culture media.

**growth factors** 

Serum-Free Medium with an Emphasis on Growth-Promoting Factors 85

parasitemia at 45 h (parasitemia-45h) were compared between parasites grown under test conditions and those grown in complete medium GFSRPMI. Different types and combinations of NEFA exerted markedly distinct effects on parasite growth in the presence/absence of phospholipids. Four typical growth patterns were defined: no inhibition (comparable to growth in complete medium); and three rate-determining steps in growth including suppressed schizogony (SS); suppressed formation of merozoites (SMF); and inhibited invasion of merozoites into new RBC (IMI)/formation of incomplete

Fig. 11. Representative modification of growth of *P. falciparum* cultured synchronously in the presence of various growth promoters, indicating comparable growth, SS, SMF, and IMI. Each developmental stage was compared with complete growth in GFSRPMI (control): ring forms (—**○**—), late schizonts (…**▲**…), parasitemia (—X—), and released merozoites (closed bars). Parasites at the ring stage (adjusted to 5.0% parasitemia) were maintained in different

**5.2 Growth-rate-determining steps in development of** *P. falciparum* **cultured in various** 

All stages of the parasite cultured in medium supplemented with NEFA (C18:1-*cis*-9 plus C16:0) in the presence of phospholipids were comparable to those grown in GFSRPMI. Medium containing C18:1-*cis*-9 and C12:0 caused parasites to accumulate in clusters of ring forms, by an SS effect. SS was also observed in the presence of C16:0 alone, C18:2 plus C16:0, C20:4 plus C16:0, or C18:1-*trans*-9 plus C16:0. Partial SS (less suppressed) was detected when the mixture of C18:1-*cis*-13 plus C16:0 was added. C18:1-*cis*-9 alone and C18:1-*cis*-9 plus C22:0 suppressed the progression of parasites to merozoites following schizont formation, by an SMF effect. SMF was also observed in parasites cultured in C18:1-*cis*-9 plus C16:0 in the absence of phospholipids, indicating that exogenous phospholipids were crucial for the development of complete merozoites. Adding C18:1-*cis*-13 plus C16:0 or C16:1 plus C16:0 to the media caused accumulation of the merozoites released from mature schizonts, but the merozoites did not invade new RBC, by the IMI effect. Partial IMI (less inhibited) was

Fig. 10. Merozoites released into surrounding medium were counted (a) and the number of merozoites decreased under the pressure of graded concentrations of chloroquine (b). The numbers of merozoites are shown per 5,000 infected RBC (b).

## **5. Differing effects of NEFA and phospholipids on intraerythrocytic growth of**  *P. falciparum* **in serum-free medium**

The efficacies of NEFA in sustaining general growth of *P. falciparum* varied markedly, depending on the type, total amount, and combinations. Certain structural characteristics of NEFA, such as carbon-chain length, degree and position of unsaturation, and isomerism were important. However, the mechanisms responsible for the different abilities of the various NEFA in the presence or absence of phospholipids, and of specific proteins such as bovine and human albumin for promoting parasite growth are unknown. Subsequent experiments therefore investigated the distinct effects of various NEFA on each developmental stage of *P. falciparum*, including schizogony, merozoite formation, and reinvasion of RBC, to provide clues to the mechanisms underlying the growth-promoting properties of NEFA.

#### **5.1 Four typical growth patterns**

To assess the effects of NEFA on each developmental stage of the parasite (schizogony, merozoite formation, and reinvasion of RBC), synchronized *P. falciparum* were cultured in the presence of phospholipids and bovine albumin, further supplemented with one or two NEFA. The distribution of the parasites among the different developmental stages was determined using flow cytometry with SYBR Green I-basic at 25 and 45 h during the first cycle of growth (Izumiyama et al., 2009). Late schizonts at 25 h (schizont-25h), released merozoites at 45 h (released merozoite-45h), new ring forms at 45 h (ring form-45h), and

Fig. 10. Merozoites released into surrounding medium were counted (a) and the number of merozoites decreased under the pressure of graded concentrations of chloroquine (b). The

**5. Differing effects of NEFA and phospholipids on intraerythrocytic growth of** 

The efficacies of NEFA in sustaining general growth of *P. falciparum* varied markedly, depending on the type, total amount, and combinations. Certain structural characteristics of NEFA, such as carbon-chain length, degree and position of unsaturation, and isomerism were important. However, the mechanisms responsible for the different abilities of the various NEFA in the presence or absence of phospholipids, and of specific proteins such as bovine and human albumin for promoting parasite growth are unknown. Subsequent experiments therefore investigated the distinct effects of various NEFA on each developmental stage of *P. falciparum*, including schizogony, merozoite formation, and reinvasion of RBC, to provide clues to the mechanisms underlying the growth-promoting

To assess the effects of NEFA on each developmental stage of the parasite (schizogony, merozoite formation, and reinvasion of RBC), synchronized *P. falciparum* were cultured in the presence of phospholipids and bovine albumin, further supplemented with one or two NEFA. The distribution of the parasites among the different developmental stages was determined using flow cytometry with SYBR Green I-basic at 25 and 45 h during the first cycle of growth (Izumiyama et al., 2009). Late schizonts at 25 h (schizont-25h), released merozoites at 45 h (released merozoite-45h), new ring forms at 45 h (ring form-45h), and

numbers of merozoites are shown per 5,000 infected RBC (b).

*P. falciparum* **in serum-free medium** 

properties of NEFA.

**5.1 Four typical growth patterns** 

parasitemia at 45 h (parasitemia-45h) were compared between parasites grown under test conditions and those grown in complete medium GFSRPMI. Different types and combinations of NEFA exerted markedly distinct effects on parasite growth in the presence/absence of phospholipids. Four typical growth patterns were defined: no inhibition (comparable to growth in complete medium); and three rate-determining steps in growth including suppressed schizogony (SS); suppressed formation of merozoites (SMF); and inhibited invasion of merozoites into new RBC (IMI)/formation of incomplete merozoites (Figure 11).

Fig. 11. Representative modification of growth of *P. falciparum* cultured synchronously in the presence of various growth promoters, indicating comparable growth, SS, SMF, and IMI. Each developmental stage was compared with complete growth in GFSRPMI (control): ring forms (—**○**—), late schizonts (…**▲**…), parasitemia (—X—), and released merozoites (closed bars). Parasites at the ring stage (adjusted to 5.0% parasitemia) were maintained in different culture media.

## **5.2 Growth-rate-determining steps in development of** *P. falciparum* **cultured in various growth factors**

All stages of the parasite cultured in medium supplemented with NEFA (C18:1-*cis*-9 plus C16:0) in the presence of phospholipids were comparable to those grown in GFSRPMI. Medium containing C18:1-*cis*-9 and C12:0 caused parasites to accumulate in clusters of ring forms, by an SS effect. SS was also observed in the presence of C16:0 alone, C18:2 plus C16:0, C20:4 plus C16:0, or C18:1-*trans*-9 plus C16:0. Partial SS (less suppressed) was detected when the mixture of C18:1-*cis*-13 plus C16:0 was added. C18:1-*cis*-9 alone and C18:1-*cis*-9 plus C22:0 suppressed the progression of parasites to merozoites following schizont formation, by an SMF effect. SMF was also observed in parasites cultured in C18:1-*cis*-9 plus C16:0 in the absence of phospholipids, indicating that exogenous phospholipids were crucial for the development of complete merozoites. Adding C18:1-*cis*-13 plus C16:0 or C16:1 plus C16:0 to the media caused accumulation of the merozoites released from mature schizonts, but the merozoites did not invade new RBC, by the IMI effect. Partial IMI (less inhibited) was

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

were found to be degenerate (Figure 13).

**6. Conclusions and future perspectives** 

**SMF effects** 

**Schizonts**

**Ring forms**

Serum-Free Medium with an Emphasis on Growth-Promoting Factors 87

Microscopic examination revealed that parasites cultured in medium containing NEFA (C18:1-*cis*-9 plus C16:0), phospholipids and bovine albumin closely resembled parasites grown in GFSRPMI. In contrast, the majority of ring forms cultured in media containing C16:1 plus C16:0 or C18:1-*cis*-13 plus C16:0 (IMI effect) for 45 h were devoid of normal structures. The majority of schizonts cultured in media containing C18:1-*cis*-9 alone, C18:1 *cis*-9 plus C22:0, C18:1-*cis*-9 plus C16:0 in the absence of phospholipids (SMF effect) for 45 h

Fig. 13. Abnormal parasites (ring forms and schizonts) grown in non-optimal culture media.

In an attempt to elucidate the mechanisms responsible for growth of *P. falciparum*, growthpromoting factors were identified and a CDM suitable for complete growth of the parasite was established. The CDM consists of paired NEFA, phospholipids with specific fatty acid moieties, and specific proteins dissolved in basal medium RPMI1640 supplemented with hypoxanthine. The most effective combination of NEFA was C18:1-*cis*-9 and C16:0. The best phospholipid crucial for serum-free culture medium supplemented with NEFA was PCdi18:1 at concentrations of 80–320 μg/ml. A simple protocol for flow cytometry with SYBR Green I was developed and used to analyze the various developmental stages of *P. falciparum*. Different stages of the parasite in RBC and released merozoites were quantified using this flow cytometry protocol. These techniques were applied to investigate the distinct roles of the identified growth-promoting factors in the development of the parasite, demonstrating that different combinations of NEFA and phospholipids exerted distinct

roles in the growth of *P. falciparum* by sustaining development at different stages.

These findings can be usefully applied in diverse aspects malaria research, including drug resistance, vaccine development, genetics, parasite biochemistry, and studies of the relationship between the parasites and the host RBC. Culture in CDM produces similar results to those using the original culture method with human serum (Trager & Jensen, 1997), with the added advantage of avoiding the adverse effects caused by human serum. In particular, the methods reported here will allow the components crucial to each developmental stage of the parasite to be established. We have already performed a large-

**5.3 Microscopic examination of** *P. falciparum* **cultured with NEFA exerting IMI and** 

**Normal Abnormal**

detected when C18:1-*cis*-9 plus C14:0, C18:1-*cis*-6 plus C16:0, C18:1-*cis*-11 plus C16:0, or C18:1-*cis*-9 plus C18:0 were added. Any effects on steps that governed parasite growth rate disrupted the cyclic behavior of the parasite, and reduced parasitemia at 45 h culture. These results indicate that different NEFA exert distinct roles in parasite development by arresting development at different stages (Figure 12).

Fig. 12. Growth-rate-determining step and growth level in development of *P. falciparum* cultured in the presence of NEFA alone or in combination. Growth of the parasite was examined in the presence of optimal phospholipids, except for C18:1-*cis*-9 plus C16:0 in the absence of phospholipids (\*) and GFSRPMI. Bovine albumin was added to the medium, except for GFSRPMI.

detected when C18:1-*cis*-9 plus C14:0, C18:1-*cis*-6 plus C16:0, C18:1-*cis*-11 plus C16:0, or C18:1-*cis*-9 plus C18:0 were added. Any effects on steps that governed parasite growth rate disrupted the cyclic behavior of the parasite, and reduced parasitemia at 45 h culture. These results indicate that different NEFA exert distinct roles in parasite development by arresting

> Growth level Ring --- Trophozoite --- Schizont --- Merozoite --- New ring

development at different stages (Figure 12).

NEFA Growth-rate

C18:1-cis-9 SMF

C18:1-cis-9+C14:0 partial IMI

C18:1-cis-6+C16:0 partial IMI C18:1-cis-9+C16:0 comparable/

C18:1-cis-11+C16:0 partial IMI

C18:1-cis-13+C16:0 partial SS, IMI

C18:1-cis-9+C18:0 partial IMI

C16:0 SS

C16:1+C16:0 IMI

C18:2+C16:0 SS

C20:4+C16:0 SS

C18:1-trans-9+C16:0 SS

Phospholipids (-)\* SMF

GFSRPMI complete growth

Fig. 12. Growth-rate-determining step and growth level in development of *P. falciparum* cultured in the presence of NEFA alone or in combination. Growth of the parasite was examined in the presence of optimal phospholipids, except for C18:1-*cis*-9 plus C16:0 in the absence of phospholipids (\*) and GFSRPMI. Bovine albumin was added to the medium,

C18:1-cis-9+C16:0,

except for GFSRPMI.

C18:1-cis-9+C22:0 SMF

C18:1-cis-9+C12:0 SS

determining step

better

#### **5.3 Microscopic examination of** *P. falciparum* **cultured with NEFA exerting IMI and SMF effects**

Microscopic examination revealed that parasites cultured in medium containing NEFA (C18:1-*cis*-9 plus C16:0), phospholipids and bovine albumin closely resembled parasites grown in GFSRPMI. In contrast, the majority of ring forms cultured in media containing C16:1 plus C16:0 or C18:1-*cis*-13 plus C16:0 (IMI effect) for 45 h were devoid of normal structures. The majority of schizonts cultured in media containing C18:1-*cis*-9 alone, C18:1 *cis*-9 plus C22:0, C18:1-*cis*-9 plus C16:0 in the absence of phospholipids (SMF effect) for 45 h were found to be degenerate (Figure 13).

Fig. 13. Abnormal parasites (ring forms and schizonts) grown in non-optimal culture media.
