Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate

CAROU, MARÍA C.; EZQUERRA RIEGA, SERGIO D.; BLÁZQUEZ-CASTRO, ALFONSO; CASAS, ADRIANA G.; BLANCO, MARÍA M.; HOROBIN, RICHARD W.; STOCKERT, JUAN C.; GARCÍA VIOR, MARÍA C.; ESPADA, JESÚS; LOMBARDO, DANIEL M.

Heliyon

Cell Press

Año: 2020 vol. 6

2405-8440

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 oftetrazolium salts into colored formazans. PMS is also currently used for cytotoxicity and viability assays of cellcultures using sulfonated tetrazoliums. Under UV (340 nm) excitation, aqueous solutions of the cationic PMSshow green fluorescence (λem: 526 nm), whereas the reduced hydrophobic derivative (methyl-phenazine, MPH)shows bluefluorescence (λem: 465 nm). Under UV (365 nm) excitation, cultured cells (LM2, IGROV-1, BGC-1, and3T3-L1 adipocytes) treated with PMS (5μg/mL, 30 min) showed cytoplasmic granules with bright bluefluo-rescence, which correspond to lipid droplets labeled by the lipophilic methyl-phenazine. After formaldehydefixation blue-fluorescing droplets could be stained with oil red O. Interestingly, PMS-treated 3T3-L1 adipocytesobserved under UV excitation 24 h after labeling showed large lipid droplets with a weak green emission within adiffuse pale blue-fluorescing cytoplasm, whereas a strong green emission was observed in small lipid droplets.Thisfluorescence 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 signifi-cantly membrane-permeant, so most PMS reduction is expected to be extracellular and associated with a plasmamembrane NAD(P)H reductase. Once formed, the lipophilic and blue-fluorescing methyl-phenazine enters livecells and mainly accumulates in lipid droplets. Overall, the results reported here indicate that PMS is an excellentfluorescent probe to investigate labeling and redox dynamics of lipid droplets in cultured cells.1. IntroductionPhenazine methosulfate (PMS) wasfirst synthesized by Kehrmannand Havas (1913) [1] from phenazine and dimethylsulfate. Later it wasapplied in dehydrogenase histochemistry [2,3,4,5], and studies on thephotosynthesis of bacteria [6].The hydrophilic cation PMS is now widely used as an intermediateredox reagent for coupling reduced dehydrogenase coenzymes to thereduction of tetrazolium salts into colored formazans [7,8,9,10,11,12,13,14]. A similar electron-transfer agent, 1-methoxy-PMS, has also beenused for revealing dehydrogenase activity in living hepatocytes [15].Other similar but seldom scarcely used redox intermediates are Meldolablue, menadione, and pyocyanin.In addition, PMS is also applied in cytotoxicity and viability assays ofcell cultures, using sulfonated tetrazoliums such as MTS, WST-1, WST-8,and XTT [16,17,18,19,20]. PMS also accelerates the reduction of MTT* Corresponding author.** Corresponding author.E-mail addresses:jcstockert@fvet.uba.ar(J.C. Stockert),dlombard@fvet.uba.ar(D.M. Lombardo).Contents lists available atScienceDirectHeliyonjournal homepage:www.cell.com/heliyonhttps://doi.org/10.1016/j.heliyon.2020.e04182Received 27 April 2020; Received in revised form 27 May 2020; Accepted 5 June 20202405-8440/©2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Heliyon 6 (2020) e04182