Optimization of Imaging Flow Cytometry for

in vitro cytotoxicity testing of drugs against
Plasmodium falciparum
Shaula Geraldino, Madel Tutor, J. Enrico Lazaro
University of the Philippines – Diliman Campus
National Institute of Molecular Biology and Biotechnology

abstract. Parameters for the Amnis FlowSight imaging flow cytometer were optimized to quantitate the median
inhibitory concentration (IC50) of drugs against the malaria parasite Plasmodium falciparum in vitro. Cytotoxicity was
performed on 96-well plates as modified from Druilhe and Gentilini (1982), using acridine orange to measure DNA. Starting
from a non-treated parasite culture previously scored for parasitemia using a RAL kit (RAL Diagnostics, France), we tried
various gating strategies involving the brightfield and the FITC channels. A sequence of gatings consisting of FOCUSED ->
SINGLE -> INTERNALIZED -> BRIGHT DETAIL INTENSITY vs INTENSITY resulted in a scatter plot with at least 3 parasitized
erythrocyte populations.
The sum of these populations corresponded to parasitemia determined by microscopy. The gating algorithm was then
applied on drug treated parasites resulting in the consistent appearance of the scatter plot and its populations. Counts for the
R2 population resulted in reproducible dose response curves. The IC50 for chloroquine and artemisinin determined from these
sigmoid curves corresponded with literature values and historical data from the laboratory. Furthermore, a small but distinct
new population of parasitized erythrocytes tagged R4 appeared consistently. We are currently investigating the hypothesis
that this population might constitute a marker for a drug endpoint, perhaps as an indicator of apoptosis.
methods.

cytotoxicity AO FlowSight Gating algorithm and
assay staining reading batch analysis

results. conclusion. Visual
examination showed that the R2
population is composed of parasitized
erythrocytes, further reinforced by
the sigmoidal shape of its dose
response curve. The other
populations did not show this trend
unless their counts are added to R2.
R1 The R1 population proximity to the
unparasitized population could mean
R2
that the data is mixed with noise,
R3 while the large and bright appearance
of R3 fluorescence suggests the
R4
presence of multinucleated schizont
stage parasites or the possibility of
multiple infection. R4 is a population
Figure 1. A gating visible only by 96 hours post-
algorithm was established synchronization of untreated
beginning with obtaining a parasites or when cultures are drug
population of focused and treated, which could be a sign of DNA
single cells. Then condensation due to apoptosis.
fluorescence internalized The gating strategy used was found
into brightfield cell images Figure 2. Time course Figure 3. Dose response to give consistent results and needs
was gated for, and a experiments on curves were generated
only to be adjusted for each
scatterplot was generated untreated parasitized for chloroquine and
experiment on the basis of controls.
using the intensity and RBC showed patterns for artemisinin using the R2 Other assays will be tested to prove
bright detail intensity each subpopulation. population because it
reproducible results. Further
features. A curve corresponded with the
experimentation will be done to
characteristic for expected trend for
identify the composition of each
parasitized cultures was parasite life cycle.
population with the hypothesis that
observed and they are the different stages of the
subpopulations were parasite life cycle or possibly
acknowledgements.
marked according to spatial
separation. We would like to thank the Philippine – apoptotic phases. More dyes -
DAPI and Hoechst – are also being
such as
California Advanced Research Institutes, the Department of Science and optimized for the same gating
Technology Science Education Institute, and the National Institute of Molecular strategy.
Biology and Biotechnology for making this project possible.