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MB Sample ID: SA226979
| Local Sample ID: | MaC1_E2_2 |
| Subject ID: | SU002395 |
| Subject Type: | Cultured cells |
| Subject Species: | Plasmodium falciparum |
| Taxonomy ID: | 5833 |
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Collection:
| Collection ID: | CO002388 |
| Collection Summary: | For metabolomics experiments, two 150 mL flasks at 6% haematocrit containing tightly synchronised parasites 28-34 hrs post-invasion (5-6 hrs rupture window), were harvested via magnet purification (Miltenyi Biotech). Infected RBC density was quantitated by flow cytometry (Tham et al., 2010) and 2 mL of 3x 107 parasites were added into the wells of 24 well microtiter plates. Parasites were incubated for 1 hrs at 37o C to stabilise the culture. Following this initial incubation, 5x IC50 of the azithromycin analogue C1 and control drugs chloroquine, dihydroartemisinin (DHA) and azithromycin were added and incubated for 2 hrs. Supernatant was removed and parasites washed twice with 800 L ice-cold 1 x PBS, with cells pelleted via centrifugation at 400 x g for 5 mins at 0o C. Cell pellets were resuspended in 200 L of ice-cold extraction buffer (CHCl3/MeOH/water (1:3:1 v/v)) containing 1 µM internal standards, CHAPS and PIPES, and then incubated on ice for 1 hrs with shaking at 200 rpm. Cell debris was pelleted with centrifugation at 14800 x g for 10 mins at 0 oC. The resulting supernatant (180 µL) was transferred to Eppendorf tubes and the remaining ~20 µL were combined to make a pooled QC sample. Extraction blank samples (without cells) were prepared alongside and samples were stored at -80 ºC until analysis. |
| Sample Type: | Blood (whole) |
