Ultraview spinning disk system

Cells were seeded in 6-well plates at 5 × 10⁵ cells/well and allowed to adhere overnight. Then the cells were treated with drugs for 24 h and were trypsinised, followed by incubation with CM-H₂DCFDA (for ROS detection), Mitotracker green (for mitochondria localisation detection) and TMRM (for MMP detection) for a designated time according to their protocols. Cells were fixed in 4% paraformaldehyde for 10 min and transferred into slides through spinning them at 800 rpm for 3 min on cytospin before DAPI staining with the nucleus. Confocal microscopy images were acquired using UltraView spinning disk system (PerkinElmer) comprising CSU-X1 spinning disk head (Yokogawa) and Volocity software.

Embryos were washed off apple juice agar plates, dechorionated in bleach, and then washed in distilled water. Live and fixed embryos were mounted on slides in voltalef oil (VWR) or DABCO (Sigma), respectively, as per Evans and colleagues, 2010 [9 (link)]. Immunostained embryos were imaged on a Nikon A1 confocal system using a 40X objective lens (CFI Super Plan Fluor ELWD 40x, NA 0.6), which was used for the migration movies in Fig 4. Aside from S2 Movie (Zeiss Lightsheet system; see S1 Methods), all other timelapse imaging and wounding was performed on a Perkin Elmer UltraView Spinning Disk system using a 40X objective lens (UplanSApo 40x oil, NA 1.3). Lower magnification images of embryos were taken using a MZ205 FA fluorescent dissection microscope with a PLANAPO 2X objective lens (Leica) or a 20x objective (UplanSApo 20x, NA 0.8) on the Perkin Elmer UltraView Spinning Disk system.

Embryos were prepared and mounted as previously described (Evans et al., 2010 (link)). Random migration was imaged using an Ultraview spinning disk system (PerkinElmer), with an image taken every 2 min for 1 hr with a z-spacing of 1 μm and approximately 20 μm deep from the ventral nerve cord using a 20x UplanSApo air objective lens (NA 0.8). Maximum projections were made for each timepoint (25 μm depth) and the centre of individual plasmatocyte cell bodies tracked using the manual tracking plugin in Fiji. Random migration speed (μm/min) and directionality (the ratio of the Cartesian distance to the actual distance migrated) were then calculated using the Ibidi chemotaxis plugin.

Raw data from Wang et al. (10 (link)) was analyzed to determine the departure dynamics of the endocytosis proteins. Briefly, spinning-disk microscopy was conducted on 4-d old epidermal cells of etiolated hypocotyls were imaged with a Nikon Ti microscope equipped with a Ultraview spinning-disk system (PerkinElmer), a Plan Apo 100× 1.45 numerical aperture (NA) oil immersion objective and a CherryTemp system (Cherry Biotech) to apply the experimental temperature conditions at either room temperature (25 °C) or 12 °C. Time lapses were collected at a frame rate of one frame per 1.174 s. The 12 °C time lapses of TPLATE-GFP and CLC2-TagRFP samples were subjected to histogram-matching bleach correction and then dynamically resliced to produce kymographs in Fiji (28 (link)). The CLC2 channel was manually screened to identify kymograph traces with a visible departure track. These selected traces were then examined to compare the departure of both channels and categized as illustrated in SI Appendix, Fig. S1.

Embryos laid on apple juice/agar plates were washed off into a cell strainer and dechorionated in 5% bleach for 1 min, followed by five washes in distilled water. Embryos were mounted in 10S Voltalef oil (VWR) as per Evans et al., 2010b (link). Live embryos were imaged using a Perkin Elmer Ultraview Spinning disk system using either a 10× air (UplanSApo 10×/NA 0.4; lateral images of stage 15 embryos to show developmental dispersal of macrophages or to quantify total number of macrophages per embryo) or 40× oil immersion (UplanSApo 40× oil/NA 1.3; all remaining live imaging) objective lens. For analysis of macrophage random migration and wound responses, the ventral surface of stage 15 embryos was imaged to a depth of approximately 20 μm with a 1 μm spacing between z-planes. Time-lapse movies were assembled from z-stacks taken every 2 min for 1 h using Volocity software (Perkin Elmer) for analysis of both macrophage random migration and wound responses. Wounding was performed using a Micropoint ablation laser (Andor) to ablate the ventral epithelium on the ventral midline in the medial-most segments of the embryo as per Evans et al. (2015) (link); the inflammatory responses of macrophages in this region were then followed for 1-h post wounding.