Orca flash 2.0 camera

Hepatocytes (2.5 × 10⁵ per well of a 6-well dish) were plated with or without 7 × 10⁴ Kupffer cells, on collagen-coated glass coverslips. Cells were fixed in 4% PFA in PBS for 1 h, followed by staining with 0.1 µg/mL LD540 [30 (link)] and 1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI). Coverslips were rinsed in water and mounted in fluorescent mounting medium (Dako, Hamburg, Germany). Epifluorescence images were acquired on an Axio Observer Z1 (Zeiss, Oberkochen, Germany) in Apotome mode with a 63×, NA 1.4 Plan-Apochromat objective and a Hamamatsu Orca flash 2.0 camera. Quantification of lipid droplet size, number and total LD540 fluorescent area was performed using Fiji [31 (link)]. If indicated, data were normalized to total number of cells using equal threshold settings for all images in one figure.

All live imaging movies were acquired on a Leica DMi8 inverted microscope with a ×63 objective (oil immersion, NA = 1.4, Hamamatsu Orca Flash 2.0 camera) and at room temperature (22–25°C). To reduce autofluorescence during imaging, the culture medium was replaced with Live Imaging Solution (Thermo Fisher Scientific A14291DJ). Culture media was not replaced for imaging cells in nocodazole, DMSO, and osmo+ to enable measurement of MT dynamics in the chosen media. For EB1-GFP imaging, an image (exposure time 500 ms) was taken every 2 s for 70–150 frames depending on sample bleaching. When imaging both EB1-GFP and Jupiter-mCherry simultaneously, one image was taken every 3 s for 100 frames (exposure 500 ms). Lamp intensity was set to the lowest level that enabled visual identification of labels.