Multispectral live-cell imaging is an informative approach that permits detecting biological processes simultaneously in the spatial and temporal domain by exploiting spectrally distinct biosensors. However, the combination of fluorescent biosensors with distinct spectral properties such as different sensitivities, and dynamic ranges can undermine accurate co-imaging of the same analyte in different subcellular locales. We advanced a single-color multiparametric imaging method, which allows simultaneous detection of hydrogen peroxide (H₂O₂) in multiple cell locales (nucleus, cytosol, mitochondria) using the H₂O₂ biosensor HyPer7. Co-culturing of endothelial cells stably expressing differentially targeted HyPer7 biosensors paved the way for co-imaging compartmentalized H₂O₂ signals simultaneously in neighboring cells in a single experimental setup. We termed this approach COMPARE IT, which is an acronym for co-culture-based multiparametric imaging technique. Employing this approach, we detected lower H₂O₂ levels in mitochondria of endothelial cells compared to the cell nucleus and cytosol under basal conditions. Upon administering exogenous H₂O₂, the cytosolic and nuclear-targeted probes displayed similarly slow and moderate HyPer7 responses, whereas the mitochondria-targeted HyPer7 signal plateaued faster and reached higher amplitudes. Our results indicate striking differences in mitochondrial H₂O₂ accumulation of endothelial cells. Here, we present the method’s potential as a practicable and informative multiparametric live-cell imaging technique.

Objective: This study aimed to investigate the individual and combined effects of retinoic acid (RA) and selenium (Se) on cell viability in SH-SY5Y neuroblastoma cells, with a focus on dose- and time-dependent responses.Materials and Methods: SH-SY5Y cells were treated with varying concentrations of RA (1, 10, or 100 μM) and Se (1, 10, or 100 μM), either alone or in combination. Cell viability was assessed using the trypan blue exclusion assay at 48 and 72 hours. Statistical analyses were performed using one-way or two-way ANOVA followed by Dunnett’s post hoc test, as appropriate.Results: Retinoic acid significantly reduced cell viability in a dose-dependent manner at both time points, with 100 μM RA causing near-complete loss of viable cells. Selenium also decreased viability, but its effect was milder and became significant only at higher concentrations. Notably, the combined application of RA and Se produced a substantially stronger cytotoxic effect than either agent alone, indicating a potential synergistic or additive interaction between the two.Conclusion: These findings indicate that the simultaneous application of RA and Se can elicit a stronger cytotoxic effect in SH-SY5Y cells than either treatment alone. The results highlight the importance of redox modulation and combination strategies in neuroblastoma cell models. Further studies are warranted to elucidate the underlying mechanisms and therapeutic potential of RA and Se co-treatment.

Objective: To investigate whether pharmacological agents associated with autophagy induction or inhibition modulate vinorelbine-induced cytotoxic responses in SH-SY5Y neuroblastoma cells.Methods: SH-SY5Y neuroblastoma cells were treated with vinorelbine, lithium chloride (LiCl), medroxyprogesterone acetate (MPA), or clomipramine, either individually or in combination. Cell viability was assessed using the Trypan Blue exclusion assay in a dose- and time-dependent manner. Treatment-induced ultrastructural alterations were examined by transmission electron microscopy after 96 hours.Results: All agents reduced cell viability in a dose- and time-dependent manner. LiCl and clomipramine produced rapid loss of viability, whereas vinorelbine and MPA caused more gradual reductions. Combination treatments, particularly vinorelbine combined with LiCl or clomipramine, resulted in markedly enhanced cytotoxic effects, reducing cell viability to below 20% at 96 hours. Ultrastructural analysis revealed distinct stress-associated morphologies: vinorelbine induced mitotic stress accompanied by secondary autophagic vacuole formation; LiCl caused extensive cytoplasmic vacuolization consistent with enhanced autophagy-associated stress; clomipramine led to pronounced vacuolation and cytoplasmic disorganization; and MPA induced mixed autophagy- and apoptosis-associated features. Combination treatments further intensified these ultrastructural alterations. Although ultrastructural data were not available for the vinorelbine plus MPA group, viability data and single-agent profiles suggest overlapping cytostatic and autophagy-associated stress responses.Conclusion: These findings indicate that agents associated with autophagy induction or inhibition can substantially influence vinorelbine-induced cytotoxic responses in SH-SY5Y neuroblastoma cells. Modulation of autophagy-related stress pathways may represent a rational strategy to enhance the cytotoxic efficacy of microtubule-disrupting agents in neuroblastoma models.