Fluorescence properties of the Na+/H+ exchanger inhibitor HMA (5-(N,N-hexamethylene) amiloride) are modulated by intracellular Ph

HMA (5-(N,N-hexamethylene)amiloride), which belongs to a family of novel amiloride derivatives, is one of the most effective inhibitors of Na+/H+ exchangers, while uneffective against Na+ channels and Na+/Ca2+ exchangers. In this study, we provided evidence that HMA can act as a fluorescent probe. In fact, human retinal ARPE19 cells incubated with HMA show an intense bluish fluorescence in the cytoplasm when observed at microscope under conventional UV-excitation conditions. Interestingly, a prolonged observation under continuous exposure to excitation lightdoes not induce great changes in cells incubated with HMA for times up to about 5 min, while an unexpected rapid increase in fluorescence signal is observed in cells incubated for longer times. The latter phenomenon is particularly evident in the perinuclear region and in discrete spots in the cytoplasm. Since HMA modulates intracellular acidity, the dependence of its fluorescence properties on medium pH and response upon irradiation have been investigated in solution, at pH 5.0 and pH 7.2. The changes in both spectral shape and amplitude emission indicate a marked pH influence on HMA fluorescence properties, making HMA exploitable as a self biomarker of pH alterations in cell studies, in the absence of perturbations induced by the administration of other exogenous dyes

Analysis of ERK3 intracellular localization: dynamic distribution during mitosis and apoptosis

Extracellular signal-regulated kinases (ERK) 1, 2 and 3 are involved in cell proliferation and differentiation, and apoptosis; although ERK1/2 have been widely studied, limited knowledge on ERK3 is available. The present work aimed at investigating ERK3 distribution during cell cycle and apoptosis in human tumor HeLa cells. The analysis performed by double immunofluorescence and immunoelectron microscopy experiments revealed that during interphase ERK3 is mainly resident in the nucleoplasm in association with ribonuclear proteins involved in early pre-mRNA splicing, it undergoes cell cycle-dependent redistribution and, during apoptosis, it remains in the nucleus in the form of massive nuclear aggregates, then moves to the cytoplasm and is finally extruded

Analysis of ERK3 intracellular localization: dynamic distribution during mitosis and apoptosis

Extracellular signal-regulated kinases (ERK) 1, 2 and 3 are involved in cell proliferation and differentiation, and apoptosis; although ERK1/2 have been widely studied, limited knowledge on ERK3 is available. The present work aimed at investigating ERK3 distribution during cell cycle and apoptosis in human tumor HeLa cells. The analysis performed by double immunofluorescence and immunoelectron microscopy experiments revealed that during interphase ERK3 is mainly resident in the nucleoplasm in association with ribonuclear proteins involved in early pre-mRNA splicing, it undergoes cell cycle-dependent redistribution and, during apoptosis, it remains in the nucleus in the form of massive nuclear aggregates, then moves to the cytoplasm and is finally extruded.</p

Reconstruction of cell distribution in 3D silicon microstructures by label-free optical detection

In this work, we report the results recently obtained with an innovative microsystem for human cell studies that combines the functionality of a highly ordered, silicon microstructure as 3D incubator with a label-free analytical approach. This system is suitable for monitoring distribution of cells, for example with a mesenchymal phenotype, that are able to stretch their bodies inside the narrow and deeply etched gaps of the 3D micromachined structure and to grow adherent to vertical surfaces. We demonstrate that the extension of the cell body inside the gaps can be recovered with a label-free optical detection method based on IR spectral reflectivity measurements performed with an all-fiberoptic configuration. © 2014 AEIT

Label-free reconstruction of cell extension grown in a 3D environment

In this work, we report the results recently obtained with an innovative microsystem for human cell analyses that combines the functionality of a highly ordered, silicon microstructure as 3D incubator with a label-free analytical approach. This system is suitable for monitoring the distribution of cells, for example with a mesenchymal phenotype, that are able to stretch their bodies inside the narrow and deeply etched gaps of the 3D micromachined structure and to grow adherent to vertical surfaces. We demonstrate that the extension of the cell body inside the gaps can be monitored with a label-free optical detection method based on IR spectral reflectivity measurements performed with an all-fiberoptic configuration

In-situ label-free optical detection of cells cultured in 3D microincubators

In this work, we show that high aspect-ratio silicon microstructures can play, at the same time, the roles of a cell-selective three-dimensional microincubator for cell culture and optical label-free transducer of cell morphology mapping. Silicon microincubators, integrating a periodic array of narrow (5-μm-wide), deeply etched (50-μm-deep) gaps separated by 3-μm-thick silicon walls, are fabricated by electrochemical micromachining (ECM) technology [1], and used for culturing several both epithelial and mesenchymal cell lines. Fluorescence microscopy analyses highlight that the microincubator shows cell-selective capabilities, being mostly cells with mesenchymal phenotype able to actively colonize the deeply etched gaps and grow attached to the vertical silicon walls [2]. The microincubator also features reflectivity spectral properties typical of one-dimensional (1D) photonic crystals (PhCs) structures in the near infrared range, with high reflectivity regions separated by deep reflectivity notches. According to 1DPhC optical properties, the presence of cells inside the gaps of the microincubator strongly affects the reflectivity signal, which can be measured in-situ with a fiber-optic setup orthogonally to the silicon wall surface (x-y plane). By spatially mapping the reflected power spectrum in the vertical x-y plane, it is thus possible to infer on the extension of cells growing into the microincubator attached to silicon walls. In particular, the intensity ratio between reflectivity maximum and minimum at two different wavelengths around 1.55 μm is closely related to the cell spreading on the silicon wall inside the deeply etched gaps of the microincubator. These results clearly envisage future in-situ label-free analyses of cellular activities involving changes in cell morphology and/or adhesion (e.g. apoptosis), in a three-dimensional environment. [1] M. Bassu, S. Surdo, L. M. Strambini, G. Barillaro, Adv. Funct. Mat., 22 (2012), 1222-1228; [2] F. Carpignano, G. Silva, S. Surdo, V. Leva, A. Montecucco, F Aredia, A. Scovassi, S. Merlo, G. Barillaro, G. Mazzini, Plos ONE 7 (2012) DOI: 10.1371/journal.pone.0048556