Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate
Juan C. Stockert,
María C. Carou,
Adriana G. Casas,
María C. García Vior,
Sergio D. Ezquerra Riega,
María M. Blanco,
Jesús Espada,
Alfonso Blázquez-Castro,
Richard W. Horobin,
Daniel M. Lombardo
Affiliations
Juan C. Stockert
Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Instituto de Investigación y Tecnología en Reproducción Animal, Cátedra de Histología y Embriología, Buenos Aires, C1427CWO, Argentina; Universidad de Buenos Aires, Instituto de Oncología “Angel H. Roffo”, Area Investigación, Buenos Aires, C1417DTB, Argentina; Corresponding author.
María C. Carou
Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Instituto de Investigación y Tecnología en Reproducción Animal, Cátedra de Histología y Embriología, Buenos Aires, C1427CWO, Argentina
Adriana G. Casas
Centro de Investigaciones sobre Porfirinas y Porfirias, Hospital de Clínicas, Universidad de Buenos Aires, CONICET, C1120AAF, Argentina
María C. García Vior
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Química Orgánica, C1113AAD, CABA, Argentina
Sergio D. Ezquerra Riega
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Química Orgánica, C1113AAD, CABA, Argentina
María M. Blanco
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Química Orgánica, C1113AAD, CABA, Argentina
Jesús Espada
Experimental Dermatology and Skin Biology Group, Ramón y Cajal Institute for Health Research, Ramón y Cajal University Hospital, 28034, Madrid, Spain; Centro Integrativo de Biología y Química Aplicada (CIBQA), Universidad Bernardo O´Higgins, Santiago, 8370854, Chile
Alfonso Blázquez-Castro
Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, 28049, Spain
Richard W. Horobin
Chemical Biology and Precision Synthesis, School of Chemistry, University of Glasgow, Glasgow, G12 8QQ, Scotland, UK
Daniel M. Lombardo
Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Instituto de Investigación y Tecnología en Reproducción Animal, Cátedra de Histología y Embriología, Buenos Aires, C1427CWO, Argentina; Corresponding author.
Natural and synthetic phenazines are widely used in biomedical sciences. In dehydrogenase histochemistry, phenazine methosulfate (PMS) is applied as a redox reagent for coupling reduced coenzymes to the reduction of tetrazolium salts into colored formazans. PMS is also currently used for cytotoxicity and viability assays of cell cultures using sulfonated tetrazoliums. Under UV (340 nm) excitation, aqueous solutions of the cationic PMS show green fluorescence (λem: 526 nm), whereas the reduced hydrophobic derivative (methyl-phenazine, MPH) shows blue fluorescence (λem: 465 nm). Under UV (365 nm) excitation, cultured cells (LM2, IGROV-1, BGC-1, and 3T3-L1 adipocytes) treated with PMS (5 μg/mL, 30 min) showed cytoplasmic granules with bright blue fluorescence, which correspond to lipid droplets labeled by the lipophilic methyl-phenazine. After formaldehyde fixation blue-fluorescing droplets could be stained with oil red O. Interestingly, PMS-treated 3T3-L1 adipocytes observed under UV excitation 24 h after labeling showed large lipid droplets with a weak green emission within a diffuse pale blue-fluorescing cytoplasm, whereas a strong green emission was observed in small lipid droplets. This fluorescence change from blue to green indicates that reoxidation of methyl-phenazine to PMS can occur. Regarding cell uptake and labeling mechanisms, QSAR models predict that the hydrophilic PMS is not significantly membrane-permeant, so most PMS reduction is expected to be extracellular and associated with a plasma membrane NAD(P)H reductase. Once formed, the lipophilic and blue-fluorescing methyl-phenazine enters live cells and mainly accumulates in lipid droplets. Overall, the results reported here indicate that PMS is an excellent fluorescent probe to investigate labeling and redox dynamics of lipid droplets in cultured cells.
