Supermacs 2 separator

iRBCs in the schizont stage contain paramagnetic iron in the hemozoin, allowing them to be separated from the initial culture using magnetic-activated cell sorting (MACS)⁵⁶ . In our experiments, the LD column (MiltenyiBiotec) and SuperMACS II Separator (MiltenyiBiotec) were used to enrich late stage iRBCs. The LD column was first inserted into the separator before rinsing with MCM. Subsequently, the culture suspension was added and subjected to the magnetic field from the SuperMACS II Separator. MCM was added to the column at least thrice to ensure that the uninfected and ring stage erythrocytes were rinsed out of the system. To collect the late stage iRBCs, the column was first removed from the separator and placed in a clean collection tube. 4 ml MCM was added twice to elute all iRBCs in the column. The resultant cell suspension was then centrifuged at 600 g for 5 min before removing the supernatant. Finally, the cell pellet was re-suspended in MCM to an appropriate concentration for experiments. The amount of late stage iRBCs obtained after enrichment is generally above 80%.

Mycoplasma-free parasites were synchronized to trophozoites using sorbitol, followed by MACS CS columns on a SuperMACS II Separator (Miltenyi Biotec) to remove uninfected RBCs. Hydrophilic parasite metabolites were extracted using 1-mL 90% cold methanol with 0.5 µM [¹³C₄, ¹⁵N₁]-aspartate (Cambridge Isotope Laboratories) as the internal standard to correct for technical variations arising from sample processing. Cell pellets were disrupted by thoroughly vortexing the sample tubes, the insoluble debris was pelleted by centrifugation (13,000 × g, 10 min), and the supernatant was collected and dried down under nitrogen gas flow. Samples were then resuspended in HPLC-grade water containing 1 µM chlorpropamide (Alpha Aesar) as an internal standard to correct for signal deviation due to instrument variance. A quality control sample consisting of all samples pooled together was created and periodically measured throughout the run to monitor signal consistency throughout the otherwise randomized run order.

Ficoll-Paque density gradient centrifugation was used to enrich hematopoietic progenitors in spleen, bone marrow and cord blood (Jaatinen and Laine 2007 ). Preparation of lineage (Lin)⁻ subsets employed MACS® magnetic bead cell separation technology according to the manufacturer’s instructions (Miltenyi Biotec, Gladbach, Germany). A cocktail of biotinylated antibodies specific for human hematopoietic lineage cells (Lineage depletion kit: Miltenyi Biotec) (10 μl per 10⁷ cells: supplemented with antibody to hCD11c) was added to cell suspensions and incubated on ice for 10 min. Antibodies were specific for hCD2, hCD3, hCD11c, hCD11b, hCD14, hCD15, hCD16, hCD19, hCD56, hCD123 and hCD235a, and bound all mature hematopoietic cells, including T cells, B cells, natural killer cells, monocytes/macrophages, granulocytes, DC and erythrocytes. MACS® anti-biotin microbeads (Miltenyi Biotec) were used for separation according to the manufacturer’s protocol. Cell separation was achieved by placing the column in a SuperMACS® II Separator (Miltenyi Biotec). The column was then washed so that flow-through cells could be collected. Flow cytometry of Lin⁻ cells was used to confirm the efficiency of depletion of ≥ 95%.