Performing Drug Assays in Plasmodium falciparum Cell Lines
Drug assays are a cornerstone of malaria research, enabling scientists to measure how well antimalarial compounds inhibit Plasmodium falciparum growth. These assays help determine drug potency (e.g., IC₅₀ values), resistance levels, and efficacy of new or existing treatments.
This guide walks through the typical steps for performing a drug assay using cultured P. falciparum parasites.
1. Preparing the Parasite Culture
Synchronized parasites (usually ring-stage) are preferred for consistency
Start with:
0.5% to 1% parasitemia
1.5% to 2% hematocrit
Use a 96-well microplate for high-throughput and replicates
Total volume per well is typically 100–200 µL
2. Drug Preparation and Plate Setup
Prepare a serial dilution of the test drug across a wide concentration range
Add diluted drug to wells (e.g., 10 concentrations, each in duplicate or triplicate)
Include:
Negative controls (no drug)
Positive controls (e.g., known drug like chloroquine or artemisinin)
3. Incubation
Incubate plates for 48–72 hours at 37°C
Maintain standard gas mixture (5% CO₂, 5% O₂, 90% N₂)
Do not disturb plates during incubation
4. Readout Methods: Measuring Parasite Growth
Choose a method to assess how much the parasite grew in each well:
a. SYBR Green I Fluorescence Assay (most common)
Add lysis buffer with SYBR Green I dye after incubation
Incubate for ~1 hour in the dark
Measure fluorescence with a plate reader (excitation: 485 nm, emission: 535 nm)
Fluorescence intensity ∼ parasite DNA content
b. [3H]-Hypoxanthine Incorporation
Add radioactive hypoxanthine at beginning of incubation
Measure uptake into parasite DNA using scintillation counter
High sensitivity, but requires radioactivity handling
c. pLDH Assay
Measures parasite lactate dehydrogenase enzyme
Colorimetric (ELISA-based)
Suitable for non-radioactive workflows
d. Microscopy
Prepare Giemsa-stained smears
Manually count parasitemia (slow, but useful for confirmation)
5. Data Analysis
Plot dose-response curves (drug concentration vs. parasite growth)
Use curve-fitting software (e.g., GraphPad Prism) to determine:
IC₅₀: Concentration that inhibits 50% of parasite growth
IC₉₀, EC₅₀ for other applications
Compare IC₅₀s to standard reference values to assess resistance
Specialized Assays
Ring-stage survival assay (RSA): Detects artemisinin resistance by exposing early rings for 6h then washing off the drug and incubating for 66h
Delayed death assay: For drugs targeting apicoplasts (e.g., doxycycline); effect seen in next parasite cycle
Checkerboard assay: Assesses drug combinations for synergy or antagonism
Summary
Need a refresher to deepen the impression? Watch the video here:
Subscribe by Email
Follow Updates Articles from This Blog via Email
No Comments