RVD involves activation of conductive K+ and Cl? channels, allowing for the escape of KCl and osmotically obligated water21
RVD involves activation of conductive K+ and Cl? channels, allowing for the escape of KCl and osmotically obligated water21. Results The cell adhesion is the result of a dynamic process related to specific interactions between the substrate surface and cell ligands and is highly depended on the cell and substrate types25. of Raman microspectroscopy to detect rapid variations of macromolecule concentration due to oxidative stress and/or cell volume changes at the single cell level. Introduction Glioblastoma multiforme (GBM) is the most frequent malignant astroglial-derived tumour in adults. The average survival rate from the time of diagnosis is less than twelve months, and even in the least Rabbit Polyclonal to TK aggressive forms, GBM causes most patients to die within two year time frame1,2. Numerous studies have focused on gaining a better understanding of different molecular mechanisms exploited by invading GBM tumour cells3C5 and in recent years there has been much interest in the use of optical tools for cancer diagnostics because of their ability to detect biochemical changes occurring at the early stages of tumorigenesis6. Aside from being one of the most invasive and deadly human malignancies, GBM is a example model of a heterogeneous cancer7,8. Its heterogeneity as well as the capacity to counteract against an hostile microenvironment, cause the conventional and targeted treatments to fail a long-term remission9,10. In order to reveal heterogeneous cell responses, analysis at the single cell level is an essential requirement11. In the last few years, there has been a rapid expansion of high throughput single cell analysis, also due to an increasing use of microfluidic devices for the total analysis of single cells12,13. At present, for single cell detection, fluorescence techniques such as Fluorescence Resonance Energy Transfer, Quantitative Time-Lapse Fluorescence Microscopy and Super-Resolution Fluorescence methods, remain the most common methods used14,15. These techniques share the limitation of their dependency on the use of probes, which can affect the cell balance and homeostasis. In this respect, it is noted that Raman microspectroscopy has been recognized as a powerful technique not solely for the single cell analysis but also for the noninvasive investigation of living cells16,17. Indeed, Pranoprofen this technique allows the assessment of the overall molecular composition of the sample without requiring cell fixation, staining or lysis. Therefore, it can represent an efficient, nondestructive tool for the analysis of single living cells and the characterization of their dynamic biochemical processes18C20. In this work, the adhesion of GBM cells to silicon substrates was evaluated and Raman microspectroscopy was used to identify molecular markers for a label-free monitoring of the dynamic stress events in single cells. The biochemical variations were induced by addition of an apoptotic inducer such as, hydrogen peroxide (H2O2), nutrient depletion or by addition of dimethyl sulfoxide (DMSO). External stimulus, like a change in Pranoprofen nutrient composition or a chemical treatment, is potentially harmful, since it can induce a cell response including various morphological and biochemical modifications, or even cell death20. Cell swelling represents a marker that occurs in response to a diversity of cellular stress, such as physical Pranoprofen damage, metabolic stress (nutrient depletion and hypoxia) and chemical stress (es. Methylmercury)21,22. Several mechanisms are involved in cell swelling such as Cl?/HCO3? and Na+/H+ exchange transport systems or ions (sodium, potassium and chloride) channels activity21. Generally, the uptake of Na+ leads to increased intracellular osmolarity and swelling. Regulatory Volume Decrease (RVD) phase based on efflux of organic osmolyte such as taurin or salt, like KCl, follows the swelling phase to restore the normal volume size23,24. RVD involves activation of conductive K+ and Cl? channels, allowing for the escape of KCl and osmotically obligated water21. Results The cell adhesion is the result of a dynamic process related to specific interactions between the substrate surface and cell ligands and is highly depended on the cell and substrate types25. For this reason, it.