Imaging Cholesterol and Lysosomes in BMdM

All fluorescence images were collected on a Zeiss 880 LSM (Carl Zeiss Microscopy, LLC., White Plaines, NY). The objective used to collect the images was a ×63 Plan Apo 1.4 NA oil immersion objective. A confocal aperture of 1 AU was used for all image acquisition. For imaging of cholesterol and lysosomes, a procedure similar to that of (Kummer et al., 2022 (link)) was used. BMdM were seeded the prior day at 1.5 × 10⁵ cells per well in a 4-well Greiner Cellview cell culture dish. The next day, BMdM were stained with LysoView 633 for 1 h at a concentration of 1x in complete media per the manufacturer’s instructions. The cells were then washed twice with PBS before being fixed for 10 min at room temperature in 4% paraformaldehyde. Again, the cells were washed twice with PBS before addition of filipin staining solution in complete media. Filipin staining solution was made by diluting a 2.5 mg/mL stock of Filipin in DMSO 1:2 with 2% BSA/PBS. BMdM were then incubated at 37°C in the dark for 2 hours. The staining solution was then removed and replaced with phenol-red free complete media. Filipin was excited with a 405-nm laser, and LysoView 633 was excited with a 633-nm laser. The laser power and detector gain were held contestant across all conditions. At least six images of each condition were acquired per experiment. Experiments were conducted over three separate days. The laser power and detector gain were held constant across all conditions. Fluorescence overlays were generated with Zeiss Zen 2.3 software.