1. AlbuMAX® I: 10% in ultrapure H2O. Filter the solution through a 0.2 μ m Steritop filter unit (for the preparation of AlbuMAX® I see Subheading 3.1).
2. Complete medium: RPMI 1640 containing 2 mM l- glutamine, 25 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) and 24 mM NaHCO3 supplemented with 0.1 mM hypoxanthine, 25 μ g/mL gentamicin and 0.5% AlbuMAX® I to constitute complete medium. Store at 4 °C.
3. Incomplete medium: RPMI 1640 w/o AlbuMAX® I and w/o hypoxanthine. Store at 4 °C (see Note 1).
4. Washed type A positive human erythrocytes with a hematocrit of 50% (see Subheading 3.2).
5. D-sorbitol: 5% in ultrapure H2O. Filter through a 0.2 μ m Steritop filter unit.
6. Cell culture dishes for suspension cell lines (10 cm × 22 mm).
7. Black 96 well microtiter plate for suspension cultures with cover plate.
8. 75 cm2 cell culture flasks.
9. 12-channel pipette.
10. Antimalaria drugs: Prepare 10 mM stocks in cell culture-tested dimethyl sulfoxide (DMSO). Store at –80 °C.
11. SYBR® Green I nucleic acid stain. Store at –20 °C (see Note 2).
12. Lysis buffer: 40 mM Tris–HCl, pH 7.5, 10 mM EDTA, pH 8.0, 0.02% saponin, 0.08% Triton X-100, SYBR® Green I 1:10,000 (see Note 3).
13. Chloroquine diphosphate salt: 10 mM stock in ultrapure H2O. Store at –80 °C.
14. 100% Methanol.
15. Giemsa solution.
16. Easycoll.
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3 Methods
1. Weigh out 50 g AlbuMAX® I in a 500 mL glass cylinder and add ultrapure H2O to 400 mL.
2. Mix vigorously. It takes about 30 min until AlbuMAX® I is dissolved.
3. Prior to further processing, check carefully that AlbuMAX® I is, indeed, completely dissolved. Add ultrapure H2O to 500 mL, mix thoroughly and filter the solution through a 0.2 μ m Steritop filter unit.
4. Make aliquots of 25 mL and freeze them at –20 °C for long- term storage.
1. Use a 500 mL bag of fresh human red blood cells (usually 300–400 mL) from one donor.
2. Make 30 mL aliquots in 50 mL sterile conical tubes.
3. Pellet red blood cells by centrifugation at 2000 × g for 10 min at RT (acceleration 9, deceleration 7).
4. Discard the supernatant and wash the red blood cell pellet with incomplete medium.
5. Repeat this washing step.
6. Determine the precise volume of the erythrocyte pellet and add the same volume of incomplete medium to resuspend the erythrocyte pellet.
7. The red blood cell solution has now a hematocrit of 50% and can be stored for up to 4–6 weeks at 4 °C.
1. Maintain P. falciparum in a continuous culture in human erythrocytes in RPMI 1640 supplemented with AlbuMAX® I as serum substitute at 37 °C under controlled atmospheric conditions of 5% CO2, 3% O2, and 92% N2 [1].
2. Cultivate the infected red blood cells in 10 cm petri dishes with a hematocrit of 5% (see Note 4). Parasitemia should be main- tained between 3 and 5% (see Subheading 3.4 for details on the determination of parasitemia).
3. Dilute parasite cultures every 48 h to a parasitemia of 0.4%. Use prewarmed complete medium and uninfected erythrocytes.
The determination of parasitemia via a thin blood film, which is described below, is schematically depicted in Fig. 1.
1. Remove the culture supernatant and resuspend the infected red blood cells in 10 mL of prewarmed complete medium.
2. Transfer 100 μ L of the suspension into a 1.5 mL microcentri- fuge tube and centrifuge for 1 min at 800 × g.
3.1 Preparation of AlbuMAX® I
3.2 Preparation of Erythrocytes from Human Blood Donations
3.3 Parasite Culturing
3.4 Determination of Parasitemia via Thin Blood Film
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3. Remove 70 μ L of the supernatant and resuspend the remain- ing cell pellet.
4. Transfer 5 μ L of the suspension to a microscope slide.
5. Approach a spreader slide to the suspension.
6. Hold the spreader slide at a 30–45° angle and push it forward smoothly.
7. Wait until the smear is air dried.
8. Dip the slide in 100% methanol in order to fix the cells to the slide.
9. Wait until the smear is air dried again.
10. Incubate the slide for 15 min in Giemsa staining solution (dilu- tion: 1:15 in ultrapure H2O) in a coplin jar.
11. Remove the slide from the coplin jar, rinse it briefly in a jet of ultrapure H2O, and let it air dry.
12. Analyze the slide with a microscope at 1000× magnification in oil immersion.
13. Count the number of infected erythrocytes in 1000 erythrocytes and determine the parasitemia by applying the formula below.
14. parasitemia [ ] % = 100 ´ number of infected erythrocytes
1000 .
Fig. 1 Preparation of a thin blood film: Schematic representation
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Parasite cultures are synchronized by sorbitol treatment according to Lambros and Vanderberg [19]. In our hands and with our cul- ture conditions, the 3D7 strain has an intraerythrocytic growth cycle of 44 h. The time values given below refer to this cycle lengths.
If a given parasite strain has a shortened or prolonged intraerythro- cytic development, these values have to be adapted accordingly.
1. Synchronize the parasite culture 44 h before the planned assay.
Parasitemia should not exceed 5%.
2. Transfer the parasite culture into a 50 mL conical tube.
3. Centrifuge the culture at RT for 2 min at 800 × g (acceleration 9, deceleration 7).
4. Remove the supernatant and resuspend the pellet in 10 mL of prewarmed 5% D-sorbitol.
5. Incubate the suspension for 15 min in a water bath at 37 °C and invert the falcon tube gently after 7 min.
6. Centrifuge the suspension for 2 min at 800 × g (acceleration 9, deceleration 7) and RT.
7. Remove the supernatant and wash the erythrocytes in 20 mL of prewarmed incomplete medium.
8. Resuspend the red blood cells in 10 mL of prewarmed com- plete medium.
9. Determine the parasitemia (see Subheading 3.4).
10. Dilute the culture with the appropriate volume of uninfected erythrocytes and complete medium to a hematocrit of 5% and a parasitemia of 2%. One culture dish will be sufficient.
11. Incubate parasite culture at 37 °C and the above-described atmospheric conditions for further 44 h before starting the assay (see Note 5). Change culture medium after 20–28 h.
The SYBR® Green I drug sensitivity assay is adapted from Smilkstein et al. [9]. This assay is performed with a cell suspension containing synchronized ring-stage parasites at a parasitemia of 0.2% and a hematocrit of 2%. Each drug is screened in triplicates in a given assay and in three independent assays (see Note 6). Standard anti- malarial drugs such as chloroquine, artesunate, or dihydroartemis- inin (DHA) serve as positive controls for growth inhibition.
1. Use a black 96 well microtiter plate (see Note 7).
2. Add 50 μ L of complete medium to each well.
3. Dedicate 8 wells for the negative control (erythrocytes w/o parasites) and 16 wells for positive control (erythrocytes with parasites w/o drug). An example on how a microtiter plate is routinely loaded is shown in Fig. 2.
3.5 Parasite Synchronization by Sorbitol Treatment
3.6 Assay
Preparation of the Cell Proliferation Assay (Standard Protocol)
Maria Leidenberger et al.
tal steps in their chronological order
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4. Prepare drug dilutions in complete medium. Dilute the drugs to their sixfold starting concentration in a 1.5 mL microcentri- fuge tube. Mix properly. The sixfold starting concentration of chloroquine and artesunate is 1458 nM and of DHA 486 nM.
5. Add 25 μ L of the sixfold starting concentration of the drug to the first row of the microtiter plate (see Fig. 2). Mix carefully by pipetting.
6. Prepare serial dilutions of the drugs (1:3) by transferring 25 μ L from row 1 to row 2 with a 12-channel pipette. Mix carefully.
Again transfer 25 μ L from row 2 to row 3 using a 12-channel pipette. Continue this procedure until all dilutions have been prepared. Discard the remaining 25 μ L from the wells of the last row after dilution has been made.
7. Prepare an erythrocyte solution with a hematocrit of 4% in complete medium and transfer it into a reagent reservoir. 6 mL is sufficient for loading of a complete microtiter plate.
8. Add 50 μ L of this solution to each well of the negative control (see Fig. 2).
9. Add a highly synchronized ring-stage culture to the erythro- cyte solution in the reagent reservoir so that the final parasit- emia will be 0.4%. The final hematocrit of this parasite solution has to be adjusted to 4%. Mix carefully avoiding air bubbles.
10. Use a 12-channel pipette to immediately transfer 50 μ L of the parasite solution to each well of positive control and of the drug dilutions. Start with the positive control and the lowest drug dilution to avoid cross-contamination.
11. Incubate the loaded microtiter plate under controlled atmo- spheric conditions at 37 °C for 72 h (see Subheading 3.3).
12. Prepare 6 mL lysis buffer per plate in a reagent reservoir and add 50 μ L to each well using a 12-channel pipette (see Note 3).
13. Lyse the parasites by incubating them in lysis buffer at RT for 1 h in the dark with constant shaking.
14. Detect the fluorescence signal using a fluorescence-activated microplate reader (top reading). Use the instrument settings with an excitation wavelength of 485 nm and an emission wavelength of 520 nm (exposure time 0.1 or 0.2 s).
In order to determine the efficacy of a drug on a defined asexual blood stage of P. falciparum, malaria parasites have to be tightly synchronized to a cycle window of ± 2 h. To determine the effect of a drug on a given parasite stage, parasites have to be synchro- nized twice within an interval of 16 h (see Fig. 3).
To perform this tight synchronization, the precise duration of the intraerythrocytic growth cycle of the respective parasite strain has to be known (Note 8). Since the drug assay is only performed 3.7 Experimental
Modification: Stage- Specific Evaluation of Drug Activity
Maria Leidenberger et al.
for the duration of the respective parasite stage, it has to be stressed that the assay outlined below is only applicable for fast-acting drugs or compounds (see also Note 9).
1. Start with a parasite culture with a high initial parasitemia (10–
15%) that mainly consists of ring-stage parasites. Ideally change medium 5–8 h prior to the first synchronization.
2. Synchronize the culture twice within an interval of 16 h (see Subheading 3.5).
3. Separate surviving ring forms and erythrocytes from parasite debris on an Easycoll step gradient after the second synchroni- zation. The step gradient is made as follows. Pour a 3 mL layer Fig. 3 Time frame of the individual asexual blood stages of the P. falciparum 3D7 strain: The 44 h intraeryth- rocytic growth cycle can divided into four stages based on morphological and biochemical criteria. During almost half of the intraerythrocytic development (ring stage (1–20 h)) the parasite displays only little measur- able metabolic activity. Protein and nucleic acid synthesis are negligible. The following stage, the trophozoite (20–26 h) is characterized by an extensive RNA and protein biosynthesis. RNA synthesis peaks during this phase of parasite development. Rings and trophozoites are mononucleated cells. DNA synthesis is initiated in trophozoites. In the subsequent schizont stage (26–36 h) 4–5 rounds of mitotic divisions take place that result in a single syncytial cell containing 8–32 nuclei. In the final segmenter stage (36–44 h) individual merozoites are formed by budding of nuclei and cytoplasmic organelles from the main parasite mass. Merozoites (about 44 h) are released, which infect fresh erythrocytes and thereby initiate a new intraerythrocytic cycle. The time spans given relate to the 44 h growth cycle of the 3D7 strain. The synchronization scheme to obtain synchro- nous parasite stages with a cycle window of ±2 h is depicted in the figure
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of 75% Easycoll in incomplete medium in a 15 mL conical tube. Resuspend the cells of a culture plate in 3 mL incomplete medium and carefully put these on top of the Easycoll layer avoiding any disturbances of this layer.
4. Centrifuge the gradient at RT for 10 min at 1450 × g in a swing-out rotor centrifuge (acceleration 7 and deceleration 5).
Parasite debris (parasites that died due to the sorbitol treat- ment) accumulates in the interphase between the incomplete medium and Easycoll layer. Erythrocytes and ring stages are found at the bottom of the gradient (see Fig. 4).
5. Aspirate the cell pellet from the bottom of the gradient with a 1 mL pipette tip. To do so move the tip through the upper layers until it touches the pellet surface. Then start with the aspiration of the pellet.
6. Transfer the obtained cell pellet into a new 15 mL conical tube and wash it 3 × with 10 mL incomplete medium to remove the remaining Easycoll.
7. Resuspend the cells in 50–60 mL complete medium and trans- fer them into a 75 cm2 culture flask (Parasitemia can be checked at this point (see Subheading 3.4). It should be around 1–2%).
8. Transfer the flask to a cell culture incubator and fix it on a slowly moving shaker with its bottleneck upwards until the newly formed merozoites have completed their invasion of erythro- cytes and developed into ring stages. This step is required to maintain synchronicity, to achieve a higher parasitemia, and to obtain erythrocytes infected with a single parasite.
9. Change culture medium 30–35 h after the second synchroni- zation step. Do not replace it earlier to avoid suction of released merozoites.
Fig. 4 Easycoll gradient to separate ring stages from parasite debris after cen- trifugation: The cell pellet at the bottom contains the desired ring-stage para- sites. Parasite debris is found in the interphase between the Easycoll layer and the incomplete medium
Maria Leidenberger et al.
10. Start with the preparations for the compound screening assay in advance (roughly 1–2 h). The assay has to be ready before the parasites at the zero position (0 ± 2 h) are entering the stage of interest (see Fig. 3 for a precise definition of the zero position).
11. According to the time schedule presented in Fig. 3, the zero position corresponds to ~46 h for trophozoite stages, ~52 h for schizonts, ~62 h for segmenter stages, and ~68 h for mero- zoites after the second synchronization step (Table 1).
12. To analyze for stage-specific effects of a given drug, the rou- tine protocol described under Subheading 3.6 has to be slightly modified. Parasites have to be plated with a parasit- emia of about 1%.
13. Fluorescence will be measured at the end of a given stage except for merozoites where the fluorescence will be deter- mined 4 h after the setup of the assay (Table 1).
1. To determine the percentage of growth inhibition, export the raw data from the plate reader to Excel.
2. Arithmetically average the growth values of the positive con- trol (parasite culture w/o drugs) and determine the mean val- ues per well.
3. Subtract the averaged values of the negative control from the mean values of the positive control and determine the standard deviation.
4. Use triplicates to calculate mean values of a given drug concen- tration. Subtract the mean value of the negative control and determine the percentage of growth inhibition.