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MB Sample ID: SA069176
Local Sample ID: | P_DHA_1 |
Subject ID: | SU001072 |
Subject Type: | Cultured cells |
Subject Species: | Plasmodium falciparum |
Taxonomy ID: | 5833 |
Genotype Strain: | 3D7 |
Subject Comments: | Malaria parasite |
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Subject:
Subject ID: | SU001072 |
Subject Type: | Cultured cells |
Subject Species: | Plasmodium falciparum |
Taxonomy ID: | 5833 |
Genotype Strain: | 3D7 |
Subject Comments: | Malaria parasite |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
P_DHA_1 | SA069176 | FL011036 | Dihydroartemisisin | drug treatment |
Collection:
Collection ID: | CO001066 |
Collection Summary: | Plasmodium falciparum (3D7 strain) parasites were cultured in vitro according to the established method (Trager and Jensen 1976, DOI: 10.1126/science.781840) with minor modifications and incubated with test compounds as previously described (Creek et al 2016, DOI: 10.1128/AAC.01226-16). Briefly, parasites were brought to a tightly synchronous life stage population (within 4 hours of the 48 hour life cycle) by treating with 5% (w/v) sorbitol twice at an interval of 14 hours, and incubated for a further 58 hours to bring all parasites to mid-trophozoite stage (27-31 hours post infection). These cells were used for drug treatment and further analysis. |
Sample Type: | Cultured cells |
Treatment:
Treatment ID: | TR001086 |
Treatment Summary: | In 96 well plates, 200 μl cultures at 7% parasitemia and 3% haematocrit were incubated with 1 µM of test compounds for a further 5 hours (32-36 hours post infection). Each compound was incubated in four replicates and untreated controls were treated with DMSO. |
Sample Preparation:
Sampleprep ID: | SP001079 |
Sampleprep Summary: | After incubation with the test compounds for 5 hours, all red blood cells were settled at the bottom of the culture wells. Culture medium was carefully removed and the metabolism of the cells was quenched by placing the plate on ice and adding ice-cold phosphate buffered saline (PBS) to the culture wells. All subsequent extraction steps were performed on ice. Cells were centrifuged for 5 min at 400g in a chilled centrifuge at 4°C. The supernatant was carefully removed and 135 µl of ice-cold methanol (containing internal standards TRIS, CHAPS, CAPS and PIPES) was added followed by rapid mixing of the cell suspension using the pipette three times. Spent media samples were also prepared by adding 10 µl of the culture supernatants to 140 µl of ice-cold methanol (containing internal standards). All samples were agitated on ice for 1 hour and then centrifuged for 10 min at 1000g in a chilled centrifuge at 4°C. The supernatant was transferred to glass vials and stored at -80°C until analysis. |
Combined analysis:
Analysis ID | AN001694 |
---|---|
Analysis type | MS |
Chromatography type | HILIC |
Chromatography system | Thermo Dionex Ultimate 3000 RS |
Column | ZIC-pHILIC (Merck Sequant) |
MS Type | ESI |
MS instrument type | Orbitrap |
MS instrument name | Thermo Q Exactive Orbitrap |
Ion Mode | UNSPECIFIED |
Units | Peak height |
Chromatography:
Chromatography ID: | CH001193 |
Chromatography Summary: | Metabolite extracts were analysed using hydrophilic interaction (HILIC) liquid chromatography (LC) and high resolution mass spectrometry on an Orbitrap system. |
Instrument Name: | Thermo Dionex Ultimate 3000 RS |
Column Name: | ZIC-pHILIC (Merck Sequant) |
Column Temperature: | 25°C |
Flow Gradient: | linear gradient -time, %B as follows: 0min- 80%, 15min- 50%, 18min- 5%, 21min- 5%, 24min- 80%, 32min- 80%. |
Flow Rate: | 300 μL/min |
Internal Standard: | internal standards (CHAPS, CAPS, PIPES and TRIS; all at 1 µM) |
Sample Injection: | 10 μL |
Solvent A: | 100% water; 20 mM ammonium carbonate |
Solvent B: | 100% acetonitrile |
Chromatography Type: | HILIC |
MS:
MS ID: | MS001569 |
Analysis ID: | AN001694 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
Ion Mode: | UNSPECIFIED |
Capillary Temperature: | 300°C |
Capillary Voltage: | +50 V |
Spray Voltage: | 4kV |
Resolution Setting: | 35000 |
Scan Range Moverz: | 85- 1275 m/z |