Quantifying Malaria Sporozoite Invasion

Confluent Hepa1-6 cells seeded in a 24-well plate were infected with 5000 freshly dissected sporozoites. After centrifugation of the plate at 400×g for 5 min for sporozoites contacting mammalian cells, monolayers were incubated at 37 °C for 3 h before washing with PBS to remove extracellular (noninvading) sporozoites. Infected cells were washed, fixed, and immunostained at different time points as described^{18 (link),65 (link)}. Fixed samples were viewed with either a Nikon Eclipse 90i fluorescence microscope equipped with an oil-immersion Nikon plan Apo ×100/NA 1.4 objective, or a Nikon plan Fluor ×60/NA 0.75 objective and a Hamamatsu GRCA-ER camera (Hamamatsu Photonics, Hamamatsu, Japan) or a Zeiss AxioImager M2 fluorescence microscope equipped with an oil-immersion Zeiss plan Apo ×100/NA 1.4 objective and a Hamamatsu ORCA-R2 camera. Optical z-sections with 0.2 µm spacing were acquired using the Volocity 6.3.1 software acquisition module (Perkin Elmer, Waltham, MA).