Celena s fluorescence microscope

BODIPY 493/503 dye (Thermo Fisher Scientific, Waltham, MA, USA) was used for staining the neutral lipid droplets (LDs), and the staining method was performed according to the manufacturer and other previous reports [33 (link)]. Fully differentiated 3T3-L1 adipocytes were incubated for 15 min at 37 °C with PBS containing 2 μM BODIPY probe. Stained cells were harvested, washed twice with PBS, and LDs intensity was quantified using CytoFLEX flow cytometry (Beckman Coulter, Inc., Kraemer Blvd. Brea, CA, USA). LD morphology was observed using a CELENA S fluorescence microscope (Logos Biosystems, Anyang, Korea) after fixing for 30 min at room temperature with 4% paraformaldehyde (PFA).

Pre-adipocytes 3T3-L1 cells were seeded on slides and cultured for 48 h. Confluent cells were treated with DMI and then cultured for 8 days, both with and without CK. Cells were washed twice with PBS, fixed with 10% formaldehyde, and then blocked with 0.2% Triton X-100 and 5% normal goat serum in PBS for 1 h. Thereafter, primary antibodies (C/EBPα and PPAR-γ) were incubated at 4 °C overnight, and Alexa 488-conjugated anti-mouse IgG or Alexa 594-conjugated anti-rabbit IgG was reacted at room temperature for 1 hour. Nuclei were counterstained using Hoechst 33342 (BD Biosciences, San Jose, CA, USA), and the stained cells were observed under a CELENA S fluorescence microscope (Logos Biosystems, Anyang, Korea). Images were analyzed using Image J software, version 1.52a (NIH, Bethesda, MD, USA).