Live Microscopy of Malaria Parasite Schizonts

Viewing chambers for live microscopy were constructed as described previously3 (link),5 (link) by adhering 22 x 64 mm borosilicate glass coverslips to microscope slides. Mature Percoll-enriched schizonts were incubated for 3-4 h at 37°C in complete medium supplemented with C2 (1 μM), then ˜5 x 10⁷ schizonts were rapidly washed twice in gassed warm complete medium lacking C2, pelleting at 1,800 x g for 1 min. The cells were suspended in 50 μl of the same medium and introduced into the pre-warmed viewing chamber on a temperature-controlled microscope stage held at 37°C on a Nikon Eclipse Ni-E wide-field microscope fitted with a Hamamatsu C11440 digital camera and Nikon N Plan Apo λ 100x/1.45NA oil immersion objective. Images (DIC alone or simultaneous DIC and fluorescence) were taken at 5-10 s intervals over a total of 20-60 min, then annotated and exported as TIFFs, AVI or QuickTime movies using Nikon NIS-Elements software.

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Thomas J.A., Tan M.S., Bisson C., Borg A., Umrekar T.R., Hackett F., Hale V.L., Vizcay-Barrena G., Fleck R.A., Snijders A.P., Saibil H.R, & Blackman M.J. (2018). A protease cascade regulates release of the human malaria parasite Plasmodium falciparum from host red blood cells. Nature microbiology, 3(4), 447-455.

Publication 2018

Eclipse Ni-E wide-field microscope C11440 digital Nikon NIS-Elements software

Cells Fluorescence G 800 Held Immersion Microscope Percoll Schizonts

Corresponding Organization : London School of Hygiene & Tropical Medicine

Other organizations : Birkbeck, University of London, Institute of Structural and Molecular Biology, MRC Laboratory of Molecular Biology, King's College London

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Variable analysis

independent variables

Incubation time of mature Percoll-enriched schizonts in complete medium supplemented with C2 (1 μM)
Washing of schizonts in gassed warm complete medium lacking C2

dependent variables

Schizont morphology and behavior observed through live microscopy

control variables

Temperature (37°C) of the viewing chamber and microscope stage
Borosilicate glass coverslips adhered to microscope slides for viewing chambers
Nikon Eclipse Ni-E wide-field microscope with Hamamatsu C11440 digital camera and Nikon N Plan Apo λ 100x/1.45NA oil immersion objective

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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