Glass bottom dish

Fully differentiated adipocytes were cultured in a glass-bottom dish (Cellvis) and incubated in phenol-red-free DMEM (imaging medium) with 100 nM TMRM (Thermo Fisher) for 30 min to indicate mitochondrial membrane potential and BODIPY 493/503 (final 5 μg ml⁻¹, Life Technology) was added to label lipid droplets for the last 15 min. Cells were then washed three times with imaging medium. Live-cell images were obtained with a Nikon A1R confocal microscope with ×100 or ×60 oil immersion objectives. For time-lapse imaging, pictures were taken every 10 min.

We used the human hepatoblastoma
HepG2 cell line (American Type Culture Collection, ATCC, Manassas,
VA, USA) and human hepatocellular carcinoma cell line Alexander (PLC/PRF/5,
ATCC, Manassas, VA, USA) in this study. Cell cultures were cultivated
in Minimum Essential Medium Eagle (BioConcept Ltd., Switzerland) supplemented
with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, US), 1% l-glutamine 100× (200 mM stock, Serana Europe GmbH, Germany),
and 1% penicillin/streptomycin (Thermo Fisher Scientific, US). Cell
cultures were kept in a humidified 5% CO₂ atmosphere at
37 °C. The culture medium (EMEM) was changed once per week. Cells
were regularly checked for common culture contamination such as mycoplasma
using the MycoAlert Detection Assay (Lonza, Switzerland). All cell
lines were authenticated by short tandem repeat (STR) DNA profiling
(ATCC, Manassas, VA, USA).
Approximately 10⁵ HepG2
and Alexander cells in a volume of 30 μL were seeded into porous
collagen scaffolds. CSs were placed in a 12-well plate. After 1 h,
the fresh medium was added to cover the whole surface of CSs. For
all utilized assays in the study, cells were cultivated in CSs for
7 days in a humidified 5% CO₂ incubator at 37 °C,
and the culture medium was changed every other day. Cells seeded on
a standard glass-bottom dish (Cellvis, Mountain View, CA, USA) with
a 35 mm diameter served as the monolayer culture (MC) model.

Cells were plated on Glass Bottom Dish (Cellvis) and were fixed with 4% paraformaldehyde for 15 min. And then, these cells were permeabilized with 0.2% Triton X-100/PBS for 10 min, before blocking with 5% skimmed milk for 1 h at room temperature. Then the nuclei were stained with Hoechst 33,342 at the appropriate dilutions. Cells were imaged on a confocal microscope (Leica).

The Drosophila embryo used in this study (Fig. 2) expressed histone tagged with EGFP (w; His2Av::eGFP; Bloomington stock #23560). The embryos were collected by putting adult flies on a grape-juice agar plate for 45 min–1 h. After incubation at 25 °C for 1 h, the embryos were attached to a glass slide with double-sided tape. We use forceps to carefully roll an embryo on the tape until the embryo dechorionated. The Dechorionated embryos were embedded in 2% low-melting-temperature agarose in a Glass Bottom Dish (35 mm Dish with 20 mm Bottom Well, Cellvis). We put the Glass Bottom Dish on the microscope stage and scan the embryo along the z-axis 4 times with a 30 µm stride, then concatenate 4 reconstructed stacks to form the volume.

HFFs containing 215430-YFP expressing parasites where washed to remove extracellular parasites, and then scraped and needle pass (23 G) to release parasites. Freshly release extracellular parasites were inoculated on pre-coated poly-l-lysine (1:10 in PBS) glass bottom dish (Cellvis) at 4 °C. The live dish was mounted on the imaging chamber of DeltaVision Core microscope (AppliedPrecision) preheated to 37° C. Images were taken every 7 seconds for a total of 10 minutes. Movie was compiled in FIJI software. For the movie used in this manuscript the images were processed to account for cell drifting using the ImageJ plugin, StackReg, for recursive alignment (http://bigwww.epfl.ch/thevenaz/stackreg/).

For live cell imaging of mCherry-Golgin45, R375A or 8-KR mutant, HeLa cells were seeded on a glass-bottom dish (35-mm diameter, Cellvis) coated with fibronectin (Millipore). After 18-h transfection, cells were imaged with a 100x objective on a Perkin Elmer UltraVIEW Spinning disk confocal microscope at 43 °C. Images were acquired every 5 min for 2 h.