Live Imaging of Parasite Egress and Invasion

Synchronous ring-stage SUB2HA3:loxP parasites were mock -or RAP-treated, then ~44 hr later schizonts were Percoll-enriched and added to fresh RBCs for 4 hr to allow egress and invasion. Without an intervening centrifugation step, a sample of the culture was supplemented with Hoechst 33342 (2 μg/ml; ThermoFisher) and Alexafluor 488 Phalloidin (diluted 1:50; ThermoFisher) and immediately applied to a viewing chamber for live imaging as described previously (Collins et al., 2013b (link)). Z-stack images were acquired at 2 μm intervals using a Nikon Eclipse Ni microscope with a 100x Plan Apo λ HA 1.45 objective and a Hamamatsu C11440 digital camera. The number of phalloidin-labelled ghosts per image was counted manually for each treatment.

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Collins C.R., Hackett F., Howell S.A., Snijders A.P., Russell M.R., Collinson L.M, & Blackman M.J. (2020). The malaria parasite sheddase SUB2 governs host red blood cell membrane sealing at invasion. eLife, 9, e61121.

Publication 2020

Hoechst 33342 Alexafluor 488 Phalloidin Eclipse Ni microscope C11440 digital camera

Camera Centrifugation Ghosts Hoechst 33342 Microscope Parasites Percoll Phalloidin Rbcs Schizonts

Corresponding Organization : London School of Hygiene & Tropical Medicine

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Variable analysis

independent variables

Treatment (mock vs. RAP)

dependent variables

Number of phalloidin-labelled ghosts per image

control variables

Synchronous ring-stage SUB2HA3:loxP parasite culture
Percoll-enrichment of schizonts
Fresh RBCs for 4 hr egress and invasion
Hoechst 33342 and Alexafluor 488 Phalloidin staining
Imaging with Nikon Eclipse Ni microscope and Hamamatsu C11440 digital camera
Manual counting of phalloidin-labelled ghosts

controls

Positive control: Not explicitly mentioned
Negative control: Mock-treated parasites

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