Orca flash 2.0 ccd camera

Bone-marrow macrophages were replated on day 4 at 20,000 or 15,000/cm² in an 8-well ibidi SlideTek chamber, for imaging at an appropriate density (approx. 60,000/cm²) on day 6 or day 7. 2 h prior to stimulation, cells were incubated for 5 min at room temperature in a solution of 2.5 ng/mL Hoechst 33342 in PBS, then BMDM culture media was replaced. This staining condition was optimized to ensure no loss of cell viability and no aberrant morphological changes over a 24 h period of imaging in the conditions described below. Cells were imaged at 5-min intervals on a Zeiss Axio Observer platform with live-cell incubation, using epifluorescent excitation from a Sutter Lambda XL light source. Images were recorded on a Hamamatsu Orca Flash 2.0 CCD camera. After the start of imaging, additional culture media containing stimulus (TNF, LPS, poly(I:C), CpG, or Pam3CSK4) was injected into the chamber in situ. We have documented the reliability of the imaging workflow by establishing that distinct biological replicates give reproducible data (Figure S1D) and that distinct imaging frames of the same well provide reproducible data (Figure S1E). All data are available at https://data.mendeley.com/datasets/6wksmvh5p4/draft?a=832656ba-2bde-40a4-8bbc-4cecb1d9543d.