Flat bottom plates

2.5 x10⁵ PBMCs in a total volume of 100 μL of AIM V media (Sigma-Aldrich) were placed per well in flat bottom plates (Thermo-Fisher). For stimulation, PBMCs were either supplemented with 20 μL of Kv or recombinant protein (20mg/ul of RPMI). Plates were incubated for 36 hrs at 37°C and 5% CO₂ and spun at 500 g for 1 min at 21°C. Supernatant was aliquoted for immediate analysis.

Human neuroblastoma SH-SY5Y cells were cultured in 2D, and single cells were prepared, followed by incubation with 2.5 μM CellTracker™ Green CMFDA (5-chloromethylfluorescein diacetate, Invitrogen, OR) and 1:1600 diluted DiD (Invitrogen, OR) to label proteins and lipids, respectively. The labeled cells were subjected to neurospheroid formation as mentioned above. After 48 h of hanging drop culture, B. mandrillaris trophozoites were added to the hanging drop and incubated for 24 h. The drops were pushed into the lower plate by adding 100 μL of culture medium. The lower plate was tilted at an angle of more than 60° for 45 min at room temperature to separate the cell debris and trophozoites into the waste well. For visualization, neurospheroids and cell debris were transferred into ultralow attachment (SPL Life Sciences, South Korea) and flat-bottom plates (Thermo Fisher Scientific, United States), respectively, for confocal imaging (Nikon Eclipse Ti, Nikon, Japan).

To ensure that the naturally acquired antibodies detected in ELISA were directed to PvRMC-RBP1 and not to the (NANP)₆ tag used for biochemical characterization, we also performed an IgG absorption ELISA protocol using a synthetic (NANP)₆ peptide. Briefly, flat-bottom plates (NUNC, USA) were coated overnight with 5 µg/mL of the peptide (NANP)₆. After washing and blocking steps, plasma from 62 randomly selected PvRMC-RBP1 IgG responders were added to the plates at a 1∶100 dilution and incubated for two hours. After incubation, plasma samples were transferred to plates coated with PvRMC-RBP1 (200 ng) and the ELISA was performed as previously described.