Cell observer widefield fluorescence microscope

Gelatin selected CS2 parasites were synchronized to 15-20 h at 1% HCT and 3% parasitemia before 5 h treatment with either 5x EC₅₀ M5717 (2.8 nM) or <0.001% DMSO (equivalent volume). Ibidi µ-Slide 0.2 channel slides that had been treated at 37°C overnight with either 100 µL chondroitin sulfate A (100 µg/mL; CSA; Sigma Aldrich) in 1x PBS, or only with 1x PBS, were washed with 1x PBS before blocking at room temperature with 1% BSA (w/v) in 1x PBS for 1 h. The slides were then flushed with warm bicarbonate-free RPMI supplemented with 5% Albumax II (RPMI Alb). Immediately before binding, the treated parasite cultures were resuspended in RPMI Alb and flowed through the channels using a syringe pump at a rate of 100 µl/min for 10 min at 37°C. The slides were then washed at the same conditions with RPMI Alb for 10 min to remove unbound RBCs. Analysis was performed by imaging 10 random fields of view (281 x 178 µm) of each condition with a 40x objective using a Zeiss Cell Observer widefield fluorescence microscope and counting bound iRBCs over three biological replicates. Counts were performed using ImageJ and results were graphed in GraphPad Prism.