A Standardized Culture Medium for Comparative Drug Efficacy Evaluation Across Plasmodium and Babesia Species

Materials and reagents

Biological materials

1. Human packed RBCs: Human erythrocytes (Group type A+ for optimal growth) obtained from American Red Cross or Interstate Blood Bank-USA, collected in citrate phosphate dextrose (CPD), washed twice with incomplete medium, and stored at 4 °C for a maximum of 4 weeks

2. Parasite species and strains:

a. Plasmodium falciparum 3D7: (MRA-102; BEI Resources)

b. Plasmodium falciparum Dd2: (MRA-150; BEI Resources)

c. Plasmodium falciparum HB3: (MRA-1227; BEI Resources)

d. Babesia duncani WA1: (NR-12311; BEI Resources)

e. Babesia divergens Rouen87: (NR-52008; BEI Resources)

f. Babesia MO1: (NR-50441; BEI Resources)

Reagents

1. RPMI-1640 (Gibco, catalog number: 11875093); store at 4 °C

2. Albumax-II (Invitrogen, catalog number: 11021045); store at 4 °C

3. L-Glutamine (Gibco, catalog number: 25030-081); store at -20 °C

4. Hypoxanthine (Sigma, catalog number: H9636)

5. HEPES (Gibco, catalog number: 15630080); store at 4 °C

6. Sodium bicarbonate (NaHCO₃) (Sigma, catalog number: S5761); store at 4 °C

7. Gentamicin (Gibco, catalog number: 15710-072)

8. DMEM-F12 (Gibco, catalog number: 11320033); store at 4 °C

9. Heat-inactivated fetal bovine serum (FBS) (Gibco, catalog number: 16140071); store at -80 °C

10. 50× HT Media Supplement Hybri-Max^TM (Sigma, catalog number: H0137); store at -20 °C

11. 100× Penicillin/Streptomycin (Gibco, catalog number: 15240-062); store at -20 °C

12. 0.008% Saponin (Sigma, catalog number: S7900)

13. 0.08% Triton X-100 (Sigma, catalog number: T8787)

14. 20 mM Tris-HCl, pH 7.5 (Sigma, catalog number: T2194)

15. 5 mM EDTA (Sigma, catalog number: E6758)

16. SYBR Green-I (Invitrogen, catalog number: S7563), diluted to 0.01% in lysis buffer; protect SYBR Green-I from light to prevent photobleaching and store at -20 °C

17. Dimethyl sulfoxide (DMSO) (Sigma, catalog number: D2650)

18. Phosphate-buffered saline (PBS) (Gibco, catalog number: 10010023)

19. Giemsa stain solution 1 (three-step fixative) (Fisher Scientific, catalog number: 22050273)

20. Giemsa stain solution 2 (three-step solution I) (Fisher Scientific, catalog number: 22050274)

21. Giemsa stain solution 3 (three-step solution II) (Fisher Scientific, catalog number: 22050275)

22. Antifolate drugs: Dihydrotriazines (51 compounds) and biguanides (28 compounds), sourced from Jacobus Pharmaceuticals, purity ≥95% (confirmed by reverse-phase HPLC)

Solutions

1. RPMI complete culture medium (see Recipes)

2. DFS20 complete culture medium (see Recipes)

3. SYBR Green-I lysis buffer (see Recipes)

Recipes

1. RPMI complete culture medium

2. DFS20 complete culture medium

3. SYBR Green-I lysis buffer

Note: Prepare the lysis buffer in distilled water and add SYBR Green-I to a concentration of 0.01%. Protect SYBR Green-I from light to prevent photobleaching.

Equipment

1. Biological safety cabinet (Sterile GARD III), Advance biological safety cabinet (The Baker Company, catalog number: SG603)

2. CO₂ incubator (Thermo Scientific, model Heracell VIOS 160i), set to 37 °C, 5% CO₂, 2% O₂, 93% N₂ for Babesia spp. and 4% O₂, 5% CO₂, 91% N₂ for P. falciparum

3. Sorvall legend XTR centrifuge (Thermo Scientific, catalog number: 75004521) with rotor for 15 and 50 mL tubes

4. Microscope (Nikon, model: Eclipse 5Oi)

5. Water bath (Fisher brand, model: FSGPD15D) Precision GP 10, set to 37 °C

6. Ultrasonic Bath (Branson Bransonic^® M Mechanical Bath, model: M2800)

7. 96-well flat clear-bottom tissue culture plates (Corning, catalog number: 3596)

8. Greiner Bio-One Cellstar 96-well black flat bottom (Thermo Scientific, catalog number: 237108)

9. Multichannel pipettor (Eppendorf, Research Plus, 10–200 μL and 5–50 μL)

10. Microplate reader: BioTek Synergy Mx, with 480 nm excitation and 540 nm emission filters (top reading mode)

11. Vortex mixer (Scientific Industries, model: Vortex-Genie 2)

12. Sterile 15 and 50 mL conical tubes (Falcon, catalog numbers: 352096 and 352070)

13. Sterile pipette tips and pipettors (10 μL, 200 μL, 1,000 μL)

14. Sterile 1.5 mL microcentrifuge tubes (Eppendorf, catalog number: 022431081)

Software and datasets

1. Microsoft Excel, for data organization and initial calculations

2. GraphPad Prism (version 9.1.2 or advanced), for data analysis, plotting the heat maps, and sigmoidal (S) inhibition curves for IC₅₀ determination with standard deviation (SD)

Procedure

Part I. Preparation of parasite cultures

Time required: 2–3 days for culture establishment and synchronization.

A. Prepare erythrocytes

1. Centrifuge A+ packed RBCs at 1,000 rcf (acceleration 9 and break 0) for 15 min at 4 °C to separate the remaining buffy coat and RBCs.

2. Wash RBCs three times with 10 mL of cold 1× PBS (centrifuge at 700 rcf for 5 min each wash).

3. Resuspend RBCs in incomplete RPMI-1640 medium to achieve 50% hematocrit (e.g., 10 mL of packed RBCs + 10 mL of DFS20) and store at 4 °C.

B. Determining initial parasitemia

1. Prepare thin blood smears from stock cultures of Babesia spp. and Plasmodium spp. strains, fix with a fixative solution (10 s), stain with Giemsa (Solution I and II) for 30 s each, wash off excess stain, and air-dry the slide.

2. Count at least 3,000 RBCs under a 100× oil immersion objective across 10 different fields on the slide to calculate the percentage of parasitemia.

3. Dilute cultures with fresh RBCs in complete DFS20 and/or RPMI-1640 medium to achieve 0.2% parasitemia at 5% hematocrit for assay setup.

4. Calculate in vitro parasitemia count as instructed in [33,35,42].

C. Maintain stock cultures

1. Hematocrit: Hematocrit (HCT) refers to the percentage of RBCs in a given volume of blood, representing the packed cell volume. Fresh human RBCs are stored at 50% hematocrit for laboratory use and maintained at 4 °C for up to two weeks. Routine asexual blood-stage cultures are maintained at 2%–5% hematocrit, depending on growth requirements.

2. Percent parasitemia: Parasitemia refers to the total number of all stages of (mixed stages) parasite-infected RBCs within a total red blood cell population in the in vitro cultures and is commonly expressed as a percentage. Routine maintenance cultures are kept at 1%–3% parasitemia to prevent overgrowth and nutrient limitation. Cultures that reach >5% parasitemia are diluted with fresh RBCs and complete medium to maintain healthy exponential growth and prevent synchronization loss.

3. Babesia duncani (WA-1), B. divergens (Rouen87), and B. MO1: Culture parasites in DFS20 medium at 5% hematocrit (A⁺ human RBCs) at 37 °C under a gas mixture of 2% O₂, 5% CO₂, and 93% N₂. Change medium daily and determine parasitemia from Giemsa-stained thin blood smears by light microscopy.

4. Plasmodium falciparum (3D7, Dd2, HB3) strains: Culture parasites in RPMI-1640 complete culture medium at 3%–5% hematocrit (A⁺ human RBCs) at 37 °C under a gas mixture of 4% O₂, 5% CO₂, and 91% N₂. Change medium daily and monitor parasitemia as above.

5. Subculture parasites every two generations and maintain parasitemia between 1% and 4% to ensure optimal growth conditions. For Plasmodium strains, synchronize cultures every 5–8 generations.

Critical:

1. Ensure that stock cultures are healthy before starting the assay. Monitor parasite growth to confirm exponential growth. B. duncani parasitemia typically doubles every 24 h, while that of P. falciparum increases by 4-fold every 48 h (one generation time is 48 h).

2. Include all three antibiotics when culturing multiple parasite species in parallel to minimize contamination risk, although gentamicin alone is sufficient for cultures.

Part II. Preparation of drug solutions

Time required: 1–2 h.

A. Prepare stock solutions

1. Dissolve compounds in 100% DMSO to make 10 mM stock solutions.

Note: Ensure complete dissolution by vortexing for 10–15 s. Store at -20 °C for long-term storage.

Troubleshooting: If precipitation occurs, warm to 50 °C and vortex again. Use an ultrasonic bath if necessary.

B. Prepare working dilutions

1. Perform serial 2-fold dilutions in complete DFS20 medium to create a 4-step or 12-step concentration range (e.g., 100, 50, 10, and 5 nM to 0.049 nM final concentration in assay wells).

2. Use a 96-well clear flat-bottom plate for dilutions to facilitate multichannel pipetting.

3. Ensure final DMSO concentration in assay wells does not exceed 0.5% (v/v).

4. Prepare working drug dilutions at 2× concentrations.

Note: Prepare fresh dilutions on the day of the experiment to avoid degradation or precipitation.

Part III. Set up the growth inhibition assay

Time required: 1–2 h for setup.

A. Prepare assay plates in DFS20 medium

1. In a 96-well flat-clear bottom plate (columns 1–12, rows A–H), add 100 μL of DFS20 medium to all wells except those designated for the initial drug dilutions (e.g., first column of triplicate wells for each drug, such as A1, B1, C1) (see Figure 2).

Figure 2. 96-well assay plate showing a schematic representation of serial dilutions of drugs, including positive and negative controls. Each well accommodates 200 μL of culture volume (Plasmodium or Babesia parasites) with drugs at various concentrations, prepared by 2-fold serial dilutions. Gradient color indicates the concentration differences in the 96-well flat clear-bottom plate.

2. Add 200 μL of each drug dilution (2× working concentration, e.g., 200 nM for a 100 nM final working concentration) to the first column of test wells in triplicate (e.g., rows A–F for two different drugs; see Figure 3).

Figure 3. Diagram of the SYBR Green I–based high-throughput screening (HTS) assay for assessing drug efficacy against the in vitro asexual life cycle stages of Plasmodium and Babesia parasites in human red blood cells (hRBCs). (A) Preparation of a 96-well clear flat-bottom drug plate with 2-fold serial dilutions in 100 μL of DFS20 medium. (B) Addition of 100 μL of hRBC-infected parasite culture to the drug plate (96-well flat clear bottom plate) using a multichannel pipette. (C) Incubation of the parasite–drug mixture in a 96-well clear flat-bottom plate at 37 °C in a chamber with 4% O₂, 5% CO₂, 91% N₂, and humidification for 72 h. (D) Transfer of incubated cultures to a black-bottom 96-well plate, followed by the addition of an equal volume of SYBR Green I lysis buffer to the parasite culture. (E) Measurement of SYBR Green I fluorescence to quantify parasite DNA content, with excitation at 480 nm and emission at 520 nm. (F) Export of fluorescence data for analysis, including generation of heat maps and determination of IC₅₀ with standard deviation values using Microsoft Excel and GraphPad Prism.

3. Perform a 2-fold serial dilution by transferring 100 μL from the first wells (2× drug concentration) to the subsequent wells in each row, continuing until the 12th well. Discard the final 100 μL from the 12th well to maintain consistent volumes.

4. Add 100 μL of DFS20 medium with 0.5% DMSO to no-drug control wells (maximum parasite growth; H1–H3).

5. Add 100 μL of DFS20 medium with 2 μM WR99210 (for Babesia spp.) or 10× IC₅₀ concentration of chloroquine (for Plasmodium spp.) to positive control wells (for 100% parasite inhibition; wells H4–H6).

Note: Ensure a uniform cell suspension by gently pipetting before dispensing. From this point forward, all parasite cultures and drug treatments are maintained in DFS20 medium.

B. Add parasite cultures to the assay plate

1. Add 100 μL of parasite culture (0.4% parasitemia, 10% hematocrit in DFS20 medium, 2× concentration) to all test wells with 2× drug concentrations. The final volume in each well should be 200 μL, with both drug and parasite at 1× concentrations.

2. Add 100 μL of uninfected RBCs (10% hematocrit in DFS20 medium) to background control wells (H10–H12).

Critical: Plasmodium cultures grown in RPMI-1640 need to be washed twice with DFS20 (700 rcf, 5 min, at room temperature). Use a multichannel pipette for consistency. Pipette slowly to avoid bubbles.

C. Incubate plates

1. Cover the plate with a low-evaporation lid to allow gas exchange.

2. Incubate at 37 °C in a humidified chamber with 4% O₂, 5% CO₂, and 91% N₂ for 72 h.

D. Measurement of parasite growth

Time required: 2–3 h.

1. Prepare SYBR Green-I lysis buffer according to the provided recipe.

2. After 72 h of incubation, remove the 96-well clear flat-bottom drug and parasite mix plate from the incubator.

3. Gently resuspend the culture in each well by pipetting up and down 5 times to ensure homogeneity.

4. Transfer 100 μL of each culture to a new black flat-bottom 96-well plate containing 100 μL of SYBR Green-I lysis buffer per well.

5. Mix gently by pipetting and incubate the plate at 37 °C in the dark (covered with aluminum foil) for 15 min to allow complete lysis and DNA staining.

6. Read fluorescence using a BioTek Synergy Mx Microplate Reader (or equivalent, e.g., Spectra Mx) with excitation at 480 nm and emission at 540 nm. (These wavelengths were routinely used and well-established to detect the SYBR Green base assessment.)

Critical: Protect SYBR Green-I from light to prevent photobleaching. Some protocols may require a 1-h incubation for complete lysis; adjust as per your experimental requirements.

Troubleshooting: If fluorescence is low, ensure thorough mixing for complete RBC lysis.

E. Data analysis

Time required: 1–2 h.

1. After completing the fluorescence reading, export the total fluorescence values from the assay to an Excel spreadsheet.

2. Each assay plate includes positive control wells (e.g., treated with WR99210 or chloroquine, CQ) and background control wells (uninfected RBCs).

3. Calculate the average fluorescence (AFU) of the uninfected RBC wells (background control). Subtract this background fluorescence value from all test wells, negative control wells (0.1% DMSO), and positive control wells to normalize the data.

4. For each test well, calculate the percent growth and inhibition using the following formulas:

% G r o w t h = ( F l u o r e s c e n c e o f t e s t w e l l - F l u o r e s c e n c e o f p o s i t i v e c o n t r o l ) ( F l u o r e s c e n c e o f n e g a t i v e c o n t r o l - F l u o r e s c e n c e o f p o s i t i v e c o n t r o l ) × 100

% I n h i b i t i o n = 100 - % G r o w t h

5. Export the % inhibition values of each drug concentration in the group format in GraphPad Prism and adjust the color format. Generate the heat maps.

6. Plot percent inhibition (y-axis) against log-transformed drug concentrations (x-axis) using GraphPad Prism.

7. Fit a sigmoidal dose-response curve (variable slope) to calculate IC₅₀ values with derived SD from biological triplicate experiments

8. Perform the analysis using data from three independent experiments, each with technical triplicates.

Critical:

1. Negative control (0.1% DMSO) represents 0% inhibition (maximum parasite growth).

2. Positive control (WR99210 or CQ-treated) represents 100% inhibition (no parasite growth).

3. Uninfected RBC wells serve as the background control for fluorescence subtraction.

Troubleshooting: If IC₅₀ curves appear irregular, verify pipetting errors or insufficient drug solubility. Remove or adjust obvious outliers from the dataset as needed.

Validation of protocol

This protocol (or parts of it) has been used and validated in the following research article:

1. Vydyam et al. [33]. In vitro efficacy of next-generation dihydrotriazines and biguanides against babesiosis and malaria parasites. Antimicrob Agents Chemother (Figure 4).

General notes and troubleshooting

1. Standardized culture medium (DFS20): Using DFS20 ensures consistent nutrient availability (putrescine, linoleic acid, lipoic acid) across Babesia and P. falciparum cultures, critical for cross-species comparisons.

2. Accurate parasitemia adjustment: Starting at 0.2% parasitemia ensures logarithmic growth during the 72-h incubation, maximizing assay sensitivity.

3. SYBR Green-I fluorescence: Thorough mixing during lysis is essential for consistent fluorescence readings. Avoid bubbles to prevent reader artifacts.

4. Once a reproducible culture system for B. microti becomes available, we anticipate that this platform can be extended to include this species and additional Babesia parasites in future studies.

Personal notes and tips

1. Culture stability: Monitor cultures daily. If parasitemia exceeds 5%, subculture to avoid stress-induced variability in drug response.

2. Compound solubility: Some compounds may exhibit limited solubility. Sonicate briefly or warm stock solutions to 37 °C to ensure complete dissolution.

3. Pipetting accuracy: Use calibrated pipettes and practice consistent technique to minimize variability in drug dilutions and culture dispensing.

Acknowledgments

The parasitology research program led by Dr. Ben Mamoun that enabled the development of this assay was supported by NIH grants AI123321, AI138139, AI152220, and AI136118, as well as by the Steven & Alexandra Cohen Foundation (Lyme 62-2020).

All laboratory assays were performed and data analysis and interpretation were conducted by Dr. Pratap Vydyam in the laboratory of Dr. Choukri Ben Mamoun.

We acknowledge our work published in Antimicrobial Agents and Chemotherapy (2024; DOI: 10.1128/aac.00423-24) [33], which served as the conceptual framework for this manuscript.

The following figures were created using BioRender: Graphical overview, BioRender.com/2q2brl8; Figure 3, BioRender.com/ nprxs56.

Competing interests

The authors declare that they have no competing financial or non-financial interests related to this work.

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