Axiocam mrn

To observe the surface biotinylation of C. sinensis, the flukes were biotinylated with 1 mM EZ-Link sulfo-NHS-SS-biotin, as described previously, washed three times in HBSS, incubated with streptavidin-FITC for 30 min at room temperature and washed three times with HBSS. Unlabeled (no biotin) flukes incubated with streptavidin-FITC alone and biotin-labeled flukes not incubated with streptavidin-FITC were used to measure the autofluorescence and as negative controls. The samples were visualized using a Zeiss Axio Imager M2 ApoTome fluorescence microscope (Zeiss, Germany) equipped with an AxioCam MRN at 40 × magnification.

The spheroids were submerged into an environment containing 10⁶ neutrophil, and time-lapse images were captured for a period of 20 h, at 10-min intervals, with camera (Axiocam mrn, Zeiss, Göttingen, Germany) attached to an inverted microscope (Axio Observer Z1, Zeiss, Göttingen, Germany). The sphere area from each time point was plotted and analyzed using ImageJ software. The growth of spheres was determined by comparing the initial and the final size of each spheroid and expressed as area (pixel²).

Cells were plated in a 6-well plate (6x10⁴ cells/well) and then treated with drugs or vehicle media for 24 h or 7 d. After the treatments, cells were detached with trypsin-0.25% EDTA, washed and plated on fibronectin-coated glass-bottomed dishes (2 μg/ml) in DMEM low glucose 10% FBS with or without drugs for 1 h, and the cells were then maintained in an incubator at 37°C. For analysis of the migration properties, phase microscopy time-lapse images were captured for a period of 20 h at 10 min intervals (migration speed and spatial trajectory (ST)) or 50 min at 10 s intervals (protrusion activity) (Ecplan-Neofluar 10x/0.3 aperture objective) with a charge coupled device camera (Axiocam mrn, Zeiss, Göttingen, Germany) attached to an inverted microscope (Axio Observer Z1, Zeiss, Göttingen, Germany) using AxioVision Software (Zeiss, Göttingen, Germany). The values for the assessment of migration speed and ST were obtained using Image J (National Institute of Health, MA, USA) software, and the data were processed as previously described [44 (link)]. For ST analysis, a polar plot graph was constructed, which represents the spatial trajectory developed by each migratory cell, where the X and Y coordinates of each cell trajectory were normalized to start at a virtual (X = 0 and Y = 0) position. A total of 80–100 cells were analyzed from 4 independent experiments for each condition.