D.A. Molina et al.
International Journal for Parasitology: Drugs and Drug Resistance 16 (2021) 129–139
per mL at 37 ◦C with 5% CO2.
medium. T. gondii tachyzoites (5 × 105) were treated for 24h directly
with molecules and the Pyrimethamine at 10 μM as a control. The par-
2.2.2. Parasites
asites that were glowing green were considered viable. The percentage
of viable cells was calculated using the following formula: (Fluorescence
of the sample with treatment URF * 100)/fluorescence of the control
without treatment, where URF = unit relatives of fluorescence calcu-
lated with the formula: (sample fluorescence/fluorescence in blank
well) *100. Parasites in 96-well plates were directly studied by fluo-
rescence microscopy by using a standard fluorescence EVOS Light Cube
GFP. Excitation 470/22 Emission: 526/60 INVITROGEN LIFE TECH-
NOLOGIES with a 10× objective on EVOS FL Color Imaging System.
Images were recorded with and adjusted for contrast with ImageJ. The
fluorescence of the wells with parasites treated as compared with those
of the control without treatment. For all the methodologies the com-
pounds were dissolved in DMSO (Fischer Scientific). The concentration
for DMSO did not exceed 1% in all assays to prevent cytotoxicity.
Toxoplasma gondii strains modified genetically that express enzyme
substrate or fluorescent labels were used for the in vitro experiments:
T. gondii RHβ1 strain (β-galactosidase tagged, kindly donated by Dr. John
Boothroyd from Stanford University), T. gondii RH GFP strain (green
fluorescent protein-tagged, kindly donated by Dr. David Sibley, Uni-
versity of Washington) and the T. gondii ME49 cherry strain (cherry
fluorescent-tagged, kindly donated by Dr. Laura Knoll, University of
Wisconsin) were maintained in DMEM media supplemented with 10%
heat-inactivated bovine serum (GE Healthcare Life Sciences).
2.3. Infection of human cell lines and measurement of the effect of
compounds on T. gondii growth
T. gondii RH-GFP and RHβ1 were passaged in confluent human
foreskin fibroblast (HFF) monolayers. RH-GFP (Green Fluorescent Pro-
tein) does not require external factors and is stable throughout a wide
spectrum of conditions (Striepen et al., 1998). RHβ1 is the RH strain
carrying the Escherichia coli lacZ (β-galactosidase) gene under the con-
trol of the SAG1 promoter (Seeber et al., 1996). To identify the growth
rates of T. gondii tachyzoites in HFF cell culture, three methodologies
were used. To establish a direct effect over the parasite and its growth
T. gondii GFP parasites of the strain RH (virulent) were used with the
ability to emit fluorescence upon receiving excitation at 395–475 nm, so
a green fluorescence can be generated and observed in a fluorescence
microscope. The other two are quantitative methodologies, the first one
using T. gondii β-Gal parasites of the RH (virulent) strain that is genet-
ically modified that constitutively express beta-galactosidase, then the
substrate X-Gal is added, producing a blue coloration that can be
quantified by spectrophotometric reading at 615 nm wavelength. The
second one is by using ME49 cherry fluorescence quantified by the
IncuCyte Performing analysis, without removing cells from the incu-
bator or disturbing cultures that automatically acquire and analyze
fluorescence and phase-contrast images around the time.
2.6. T. gondii RH GFP tachyzoites in 48h growth assay
HFF cells were plated in 96 well plates at approximately 20,000 cells
per well and allowed to grow until confluent. Parasite cultures that had
completely lysed an HFF monolayer were recollected into a centrifuge
tube and were washed with 10 mL of PBS 1X centrifuging to 500 g × 5
min at room temperature to remove host cell debris and parasite ag-
gregates. The supernatants were passed to another centrifuge tube and
the pellets were collected after centrifuging to 1,800 g × 10 minutes at
room temperature. The resulting parasites were resuspended, counted,
and diluted in media to a concentration of 2 × 106 per mL, and 5 μL of
this suspension was added to the appropriate wells of the microtiter
plates, which already contained the compounds to be tested, to give an
inoculum of approximately 10,000 parasites per well then we added the
molecules. For the assays with the RH GFP strain, the fluorescence was
quantified by fluorescence microscopy using a standard fluorescence
EVOS Light Cube GFP. Excitation 470/22 Emission: 526/60 INVI-
TROGEN LIFE TECHNOLOGIES with a 10× objective on EVOS FL Color
Imaging System. Images were recorded with and adjusted for contrast
with ImageJ. The plates were then incubated at 37 ◦C with 5% CO2 for
48h. After this time each well was gently washed and media replaced.
The fluorescence of the well with parasites treated was compared with
the control without treatment. GFP fluorescence was quantified by
fluorescence microscopy using a standard fluoresce EVOS Light Cube
GFP. Excitation 470/22 Emission 526/60 INVITROGEN LIFE TECH-
NOLOGIES with a 10× objective on EVOS FL Color Imaging System.
Images were recorded with and adjusted for contrast with ImageJ. The
plates were then incubated at 37 ◦C with 5% CO2 for 48h.
2.4. HFF cell viability by Alamar blue assay
HFF cells were cultured as monolayers and maintained in Dulbecco’s
modified Eagle medium (DMEM) supplemented with 10% fetal bovine
serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin in a
humidified atmosphere with 5% CO2 at 37 ◦C in T-25 flasks and were
subcultured twice a week. Cell lines were plated with approximately
20,000 cells per well and let grown until confluence. Once confluent, the
cells were treated with a compound concentration of 10 μM for 24 h.
This was followed by an Alamar Blue assay with 0.5 mM resazurin at
37 ◦C with 5% CO2 for 4 h. All concentrations were performed in trip-
licated. A fluorescent reading was then taken with a BioTek Synergy HT
plate reader at 530/25 excitation and 590/25 emission. Host cell
viability was determined by comparing the treatments with no com-
pound treatment as 100% viability. A color change from blue to pink
indicated the metabolically active cells. A color change in the growth
control well to pink indicated proper cell growth and no color change in
the sterile control well-indicated absence of contaminants.
2.7. T. gondii expressing β-galactosidase for colorimetric assessment
We used microtiter assay for drug evaluation with a strain of T. gondii
that expresses bacterial β-galactosidase. Parasites were kindly donated
by John Boothroyd. This assay provides a high-throughput and nonra-
dioactive alternative for the identification of anti-T. gondii compounds.
An inoculum of approximately 10,000 parasites per well was used in
wells that already contained the compounds, compared with the control
without treatment. The plates were then incubated at 37 ◦C with 5% CO2
for 96 h. After this time 2 μl of X-Gal 30 mg per mL (Sigma-USA), was
2.5. T. gondii RH GFP tachyzoites 24h viability assay
added to give a final concentration of 100 μM of X-gal. The plates were
incubated at 37 ◦C with 5% CO2 for an additional 24 h and were then
read at 420 and 615 nm on a Bio-Tek microtiter plate reader. Note that:
RH-strain parasites were grown in HFF cells. Parasites to be used in
the assay were prepared as follows. Cultures that had completely lysed
an HFF monolayer were recollected into a centrifuge tube and were
washed with 10 mL of PBS 1X centrifuging to 500 g × 5 min at room
temperature to remove host cell debris and parasite aggregates. The
supernatants were passed to another centrifuge tube and the pellets were
collected after centrifuging to 1800 g × 10 min at room temperature.
The resulting parasites were resuspended, counted, and diluted in the
● Previously a suitable number of parasites was standardized to
perform the experiments since this number allowed to find differ-
ences in the readings and coloration produced by the X-Gal substrate.
Thus, for each experiment 10,000 parasites per well were used in
plates of 96 well to perform the HFF cell growth experiments after
adding 4-thiazolidinone molecules.
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