Nile red

Differentiated 3T3-L1 cells were carefully washed twice with Dulbecco’s phosphate buffered saline (DPBS), fixed in 3.7% formalin for 30 min, washed twice again in DPBS, and stained with 0.5-μg/mL Nile Red (Cayman Chemical, Ann Arbor, MI) and 1-μg/mL Hoechst 33342 (Invitrogen, Carlsbad, CA) for 10 min, as described earlier [22 (link)]. The lipid droplet staining and cell morphology were examined using a fluorescence microscope (NIB410, Nexcope, Ningobo, China) at ×200 magnification. All images were analyzed using ImageJ (NIH, Bethesda, MD).

The following reagents were purchased from Cayman Chemicals - u18666A (cat #10009085), ketoconazole (cat #15212), Filipin III (cat #70440), NileRed (cat # 30787). Gemcitabine (cat #G6423) and cholesterol (cat #C8667-1G) were purchased from Sigma.

To visualize the localization of BODIPY^TM FL C12 and its incorporation into neutral lipids in microglia treated with FFAs, microglia cells were seeded on glass coverslips in 24-well plates at 2.5 × 10⁴ cells/well, incubated overnight, and treated with 200 µM FFAs or BSA in serum-free media for 6 h. Following treatment, cells were incubated with 15 μM BODIPY^TM FL C12 (ThermoFisher Scientific; Waltham, MA, USA) and 2 μM Nile Red (Cayman Chemical; Ann Arbor, MI, USA) in serum-free media for 30 min at 37 °C. Cells were then rinsed with D-PBS, fixed with 4% paraformaldehyde for 20 min at 4 °C, rinsed with D-PBS again, and stained with 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) in D-PBS for 5 min. Cells were then rinsed with D-PBS again, and then the coverslips were mounted onto glass slides and visualized under confocal microscopy.