For ex vivo drug sensitivity testing, a semi-automated radioactive microplate method was used to measure the IC50 as described in a previous study [33 (link)]. Briefly, at day 0, ring-form parasites obtained after blood collection were washed three times in RPMI 1640 media. Infected Red blood cells were suspended in 10% human serum completed culture media. After adjustment of parasitaemia between 0.5 and 1% and hematocrit at 1%, an equivalent of 1 μ Ci per well of tritiated hypoxanthine with a specific activity of 14.1 Ci/mmol (Perkin-Elmer, Foster City, CA) was added in a parasite suspension. A final volume of 200 μl per well of parasite suspension was placed into drug-prefilled 96-well tissue culture plates. The parasite suspension was mixed and incubated with each drug at various concentrations of drug at 37 °C with 5% CO2, 5% O2 and 90% N2 for 42 h. After one step of freezing and thawing, the parasites were collected on filter plate, dried and 50 μl of scintillation cocktail (Optiphase Supermix; Perkin-Elmer) was added on each well. Tritium incorporation was determined with a beta-counter (Wallac 1450 microbeta trilux; Perkin-Elmer), and the IC50 was determined after the drug concentration was plotted against the radioactivity by the online IC50 estimator [34 ]. Three wells without drug for each concentration panel and the reference clones 3D7 and Dd2 were used as control.
Tritiated hypoxanthine
Manufactured by PerkinElmer
Sourced in Finland, France
Tritiated hypoxanthine is a radiolabeled compound used in various research applications. It serves as a precursor for nucleic acid synthesis and can be used to measure DNA and RNA turnover in biological systems. The tritium label allows for the detection and quantification of the compound during experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Wallac 1450 microbeta trilux, by PerkinElmer (1 mentions)
Optiphase supermix, by PerkinElmer (1 mentions)
Wallac microbeta trilux, by PerkinElmer (1 mentions)
Unifilter 96, by PerkinElmer (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Unifilter 96 gf b pei coated plates, by PerkinElmer (1 mentions)
4 protocols using tritiated hypoxanthine
1
Ex vivo Drug Sensitivity Assay
2
In Vitro Drug Sensitivity Assay for Malaria Parasites
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Drug sensitivity tests were performed within 24 hours of bleeding, without culture adaptation. Blood samples were washed three times in RPMI 1640 medium (Sigma Aldrich, St. Louis, USA). The modified isotopic microtest technique [14 (link)] was used. The complete RPMI 1640 medium, i.e. supplemented with 10% of human serum type AB (obtained from the blood bank of Souro Sanon University hospital, Bobo-Dioulasso, Burkina Faso), gentamicin (10 μg/ml) and tritiated hypoxanthine (Amersham, little Chalfont, UK), was used for parasites cultivation. Infected erythrocytes were suspended in this medium at a haematocrit of 1.5% and an initial parasitaemia between 0.1% and 0.5%. This suspension was then added in quantities of 200 μl per well to the plates containing the drugs. These were incubated for 48 hours at 37°C in 5% CO2. After the incubation period, plates were frozen and thawed to lyse the blood cells. Cultures were then harvested (Harvester Unifilter 96, Packard) on fibre glass paper (reader plates Unifilter 96 Perkin Elmer). The strips obtained were dried and transferred to a plastic bag containing 30 μl of scintillation fluid (Perkin Elmer Betaplate Scint, Wallac). The incorporation of tritiated hypoxanthine was measured using a scintillation Beta counter (Perkin Elmer Wallac MicroBeta Trilux, Turku, Finland).
3
Antiplasmodial Activity Evaluation
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The antiplasmodial activity was assessed on the chloroquine-resistant Plasmodium falciparum strain FcM29-Cameroon, cultured continuously according to the method of Trager and Jensen (Trager and Jensen, 1976) , in a 5% CO 2 atmosphere at 37°C as previously described (Benoit-Vical et al., 2007) (link). Briefly, the parasites were maintained in vitro in human red blood cells and diluted in RPMI 1640 medium, supplemented with 25 mM HEPES, 2.05 mM L-glutamine and completed with 5% human serum (French Blood Bank, EFS). The antiplasmodial activity was assessed as previously reported by Desjardins et al. (Desjardins et al., 1979) (link) and modified as follows. Extract dilutions and compounds were tested 3 times independently, each dilution in triplicate, in 96-well plates with cultures at a parasitaemia of 1% and a haematocrit of 1%. For each test, the plates of parasite culture were incubated with products for 48 h and tritiated hypoxanthine (Perkin Elmer, France) was added to the medium 24 h after the beginning of incubation (Benoit-Vical et al., 2007) (link). The parasite culture control (with solvent only) was referred to as 100% growth. Parasite growth was estimated by [ 3 H]hypoxanthine incorporation. Inhibitory concentration 50% (IC 50 ) was defined as the concentration of drug required to inhibit 50% of the metabolic activity of Plasmodium falciparum compared to the control.
4
Malaria Drug Susceptibility Assay
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One hundred mL of rings at 1% parasitemia and 2.5% hematocrit in RPMI were dispensed in a 96-well round bottom plate. Then, 100 mL of drugs (4.0-0.003 mM) previously suspended in RPMI was dispensed in the respective wells. Each drug was tested in triplicate, in seven different concentrations. The untreated parasite samples received 100 mL of medium containing 0.5% DMSO. Chloroquine was used as a positive control. The plates were incubated for 24 h at 37 1C under 3% O 2 , 5% CO 2 and 91% N 2 atmosphere. Then, 25 mL of tritiated hypoxanthine (0.5 mCi per well, PerkinElmer, Shelton, CT) in RPMI was added to each well and incubated for 24 h. The plates were frozen at À20 1C and subsequently thawed and the contents transferred to UniFilter-96 GF/B PEI coated plates (PerkinElmer) using a cell harvester. After drying, 50 mL of scintillation cocktail (MaxiLight, Hidex, Turku, Finland) was added in each well and sealed and the plate was read in a liquid scintillation microplate counter (Chameleon, Turku, Finland). The % of inhibition was determined in comparison with untreated cells and the inhibitory concentration for 50% (IC 50 ) values were determined by using non-linear regression with the Logistic equation available in OriginPro 8.5. Three independent experiments were performed.
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