Macs cell separation ld columns

Briefly, sorbitol-synchronized iRBCs were transferred to MACS cell separation LD columns (Miltenyi Biotec, Bergisch Gladbach, Germany), washed, and eluted in CM. For measurements using live-cell microscopy, cells were washed and resuspended in Ringer’s solution (122.5 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl₂, 0.8 mM MgCl₂, 11 mM D-glucose, 25 mM HEPES, and 1 mM NaH₂PO₄, pH 7.4) to a density leading to approximately 30 parasites per field of view. For plate reader measurements, cells were counted using the improved Neubauer hemocytometer (Brand GmbH, Wertheim, Germany) and diluted to the desired parasite count.

VAR2CSA-expressing CS2 Pf⁴¹ (link) parasites were obtained through BEI Resources (NIAID, NIH: Plasmodium falciparum, Strain CS2, MRA-96, contributed by Stephen J. Rogerson.) CS2 parasites were cultured in RPMI 1640 with 25 mM HEPES, 25 mM sodium bicarbonate, 1% gentamycin, and enriched with 0.5% Albumax (pH 6.75) and 250uM hypoxanthine, under atmospheric conditions (5% oxygen, 5% carbon dioxide, and 95% nitrogen). To retain synchronous cultures, parasite growth was treated with 5% D-sorbitol. Schizont isolation was performed at 5–10% parasitaemia using MACS cell separation LD columns (Miltenyi Biotec) and cryopreserved in glycerolyte prior to use. For functional experiments, schizonts were thawed, washed, and resuspended in RPMI before addition to cells. Mycoplasma contamination was assayed using the MycoAlert Mycoplasma Detection Kit (Lonza).