Ti microscope

We performed in situ RNA hybridization following the protocol described previously [21 (link)]. Tissues were obtained from developing spikes of diploid T. monococcum (accession PI 167615) and tetraploid wheat cultivar Kronos. cDNAs obtained from T. monococcum and Kronos were used to amplify WAPO1 genes for the in vitro transcription reaction. We designed wheat A-genome specific primers appended with promoter sequences of T3 and T7 (S1 Table). Probes were synthesized using T3 (sense probe) or T7 (antisense probe) RNA Polymerase (Roche) and labelled with Digoxigenin-11-UTP (Roche). The forward primer P3-WM-APO1-T3-F₁₄₀₀ starts 57 bp upstream of the stop codon and two alternative reverse primers P4-WM-APO1-T7-R₁₆₄₉ and P5-WM-APO1-T7-R₁₈₄₃ end in the 3’ UTR and include a total of 266 and 458 bp respectively (S1 Table). The P3-P4 and P3-P5 probes showed the same specificity. The color reaction was stopped at 48 or 72 hours, and images were taken by Nikon Ti Microscope equipped with a DS-Fi2-U3 camera.

Jurkat cells were washed with PBS three times and re-suspended in PBS at a concentration of 2 × 10⁵ cells mL⁻¹. Cells were transferred to solutions with the following conditions: buffer alone, NEU3 (0.01875 U), or NanI (0.01875 U). All samples had a final concentration of 10% NEU3 storage buffer and 0.6% binding buffer (100 mM CAPS, 0.15M NaCl, 1 mM calcium chloride, pH 11.0). All samples were stained with 1 μg mL⁻¹ Calcein AM (Life Technologies, Burlington, ON, Canada). Samples were transferred to a 96-well- plate (200 μL per well). The plate was incubated at 37°C for 3 h. Fluorescent images were taken with a NIKON Ti microscope using a 20x objective and a FITC filter set. Four images were taken for each well to provide 24 images for each condition. The images were analyzed with CellProfiler (Version 2.1.1) (Carpenter et al., 2006 (link); Bray et al., 2015 (link)). The total number of the cells in each image, and the number of single cells (cells not in any clusters) were counted in CellProfiler. The amount of aggregation was calculated as (total number of cells – numbers of single cells)/total number of cells. Results were confirmed using at least two independent repeats.

To visualize GFP-tagged proteins, cells from overnight cultures were back-diluted 50× into fresh SC medium and incubated at 30 °C and 200 r.p.m. for 4–6 h. Cells were washed with water and mounted unfixed on microscope slides. Images were captured using a Nikon Ti microscope with a 100× PlanAPO lens (NA 1.49). Cells were illuminated using high inclination laminated optical sheet TIRF illumination with 488 nm lasers, and its respective filter cube (Chroma). Images are of single planes. Image processing was done using Fiji (NIH).

Glass bottom culture dishes (35 mm) were coated with 0.1% poly-l-lysine overnight and washed 3 times with PBS. Parasite culture (250 μL at 2.5% hematocrit) was added to the dishes and incubated for 30 min to allow attachment. Culture dishes were washed 3 times with PBS followed by addition of medium. For MβCD extrusion experiments with the NF54 Rhoph2/Exp2 line, all washes, incubation, and imaging were done in medium supplemented with TMP. Parasites were attached to poly-l-lysine-coated plates and incubated in RPMI containing wheat germ agglutinin-Alexa 350 (WGA-350) (Invitrogen; catalog no. W7024) at 5 μg/mL final concentration and incubated for 12 min to stain the erythrocyte plasma membrane. In addition to WGA staining, the PfVP1-mNG line was also stained with SYTO deep red nuclear stain for 30 min. Parasites were treated with 5 mM MβCD followed by three washes with medium. The medium was replaced with phenol red-free medium followed by live fluorescence microscopy. Imaging was done using the Nikon Ti microscope. WGA-Alexa 350 was visualized using a DAPI (4′,6-diamidino-2-phenylindole) filter set, mNeonGreen with (fluorescein isothiocyanate) FITC, and SYTO deep red nuclear stain with Cy5, and mRuby was visualized using tetramethyl rhodamine isocyanate (TRITC) filter set. Video microscopy parameters are given in the legends of movies in the supplemental material.