CONICET | Buscador de Institutos y Recursos Humanos

Erythrocytes are widely used as a model system for membrane studies due to their relatively simple structure (they lack nuclei and organelles having only the plasma membrane), their convenient experimental manipulation and availability. However, the high hemoglobin content inside the red blood cells may be a problem for the use of fluorescent membrane probes. Hemoglobin content inside erythrocytes is around 20 mM and the molecules near the inner membrane surface would be the responsible for the quenching of fluorescent dyes such as 1,6-diphenyl-l,3,5-hexatriene (DPH) (1) and 12-(9-anthroyl) stearic acid (AS) (2). Laurdan (6-lauroyl,1-2-dimethylamino naphthalene), a fluorescent probe commonly used to study membrane structure is being used in studies in whole erythrocytes successfully. However, in order to avoid data misinterpretation due to the quenching effect of hemoglobin, the authors either work at constant hematocrit (constant hemoglobin concentration), they apply correction factors for the energy transfer, or they work with hemoglobin depleted erythrocytes (ghosts). Taking this into account, the aim of In this work was to we explored the use of Laurdan in the presence of Hhemoglobin and during hemolysis.We studied the changes promoted in the membrane of rabbit red blood cells by the sucrose monoester of myristic acid, -D-fructofuranosyl-6-O-myristoyl-α-D-glucopyranoside (MMS). We followed the interaction before and after hemolysis using FLIM (phasor plots) and Spectral imaging (spectral phasor). Our data indicate that at sublytical concentration of surfactant (20 μM MMS), there is a decrease of about 35% in erythrocytes size, without changes in Laurdan lifetime or emission spectra. As hemolysis progress, Laurdan lifetime data are not informative due to the presence of hemoglobin but Laurdan spectral phasor analyses clearly show an increase in membrane fluidity promoted by MMS.