In vitro luciferase assays for liver stage egress, detached cell formation and merozoite numbers

In order to investigate detached cell formation by PbmCherry_Hsp70NLuc_ef1α (PbNLuc) parasites, HepG2 cells were infected as previously described, and at 63–65 h post-infection, the supernatant of the 24 well-plates, where detached cells (representing successfully completed liver stage development) are located, was collected for downstream analysis. For collection of single detached cells, the method previously published by Stanway et al. [100] was used. Prior to collection and lysis, individual detached cells were imaged by fluorescence using an inverted fluorescence microscope (Leica DMI 6000B with an incubation chamber), and a series of 3–4 z-stacks obtained for measurements. Upon collection, individual detached cells were lysed in 1xPLB, and assayed by luminescence. For consistent and unsaturated measurements, the optimal NanoGlo™ luciferase assay substrate dilution used for probing detached cells was determined to be 1:250. In order to determine the number of merozoites per individual detached cell, a previously published method [101] was used. Namely, from the fluorescence images, the radius of the detached cell was determined, and the volume of the detached cell (V_DC) was calculated by the formula VDC=43πr3. Sturm et al. previously determined the volume of individual merozoites (V_m) to be equal to 2.66 μm³ [101]. An approximation of the number of merozoites (N_m) per detached cell is, therefore, calculated as Nm=VDCVm.