Bodipy c16

After isolation, 1e6 cells from tissue single-cell suspensions were plated in a non-treated V-bottom 96 well plate. Cells were washed once with pre-warmed base RPMI and then suspended in 200ul of RPMI containing 0.5 mM BSA (Sigma, BSAV-RO), 5uM BODIPY C16 (ThermoFisher, D3821), 5 nM TMRM (ThermoFisher, M20036) and 20 nM MitoTracker DeepRed (ThermoFisher, M22426) for 20 min at 37 °C. Cells were washed 2x in cold PBS/2%FBS/2 mM BSA before lived/dead and surface staining and acquired immediately afterwards.

To monitor lymphatic function in the small intestine, mice were fasted overnight followed by feeding them with 100 μl of 4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid (BODIPY C₁₆) (Thermo Fisher, D3821) diluted in sunflower oil (Sigma-Aldrich, S5007) at 1:250. Two hours later, mice were sacrificed and imaged by fluorescent stereo microscopy.

Monolayers of macrophages infected at an MOI of 3:1 in 8-well glass bottom μ-Slides (Ibidi) were pulsed with 8 μM Bodipy-C16 (Thermo Fisher Scientific) complexed to fatty acid–free BSA as described (8 , 12 , 46 ). Following the pulse labeling period, the cells were chased in fresh media without label for 1 h. The infected macrophages were imaged by confocal microscopy as described (47 (link)).

Mouse primary hepatocytes were exposed to 0.2 mM palmitate (PA) with or without Alisol B for 48 h. After being fixed in 4% paraformaldehyde for 30 min, cells were stained with Oil Red O (Cat#71029781, Sinopharm Chemical Reagent Co., Shanghai, China) working solution for 20 min and subsequently stained with hematoxylin solution for 3 min. The respective images of Oil Red O staining were captured with an Olympus IX73 microscope at 200× magnification. To evaluate hepatic fatty acid uptake, mouse primary hepatocytes were treated with or without Alisol B for 48 h and then incubated with 100 nM BODIPY-C16 (Cat#3821, Thermo Fisher Scientific, Waltham, MA, USA) for 5 min. After being fixed with 4% paraformaldehyde for 30 min, the hepatocytes were incubated with DAPI to stain nuclei for 3 min, and the images of BODIPY-C16 fluorescence were captured at 600× magnification with an Olympus FV1000-SIM microscope.

SNB19 cells were seeded in four-well chamber slides and infected as described above. The cells were then incubated with 50 nM Bodipy-C16 (Thermo Fisher Scientific) in PBS for 15 min at room temperature. Subsequently, the samples were fixed with 4% paraformaldehyde in PBS, counterstained with DAPI, and imaged using a FSL confocal microscope (Olympus).