br Acknowledgements This work was partially

Acknowledgements This work was partially funded by Italian Ministry of Education, University and Research (MIUR) by means of the national Program PON R&C 2007-2013, project “Hyppocrates–Sviluppo di Micro e Nano-Tecnologie e Sistemi Avanzati per la Salute dell\'uomo (PON02 00355)”. Authors also gratefully acknowledge A.B.A.L. onlus Messina (Italy) (http://abalmessina.it/) for the use of the XploRA Raman spectrometer and Prof. Franco Felici for the kind gift of clone selection from his phage-display libraries.
Introduction Dielectrophoresis (DEP) is currently used to determine the electrical characteristics of the cells, which are then analyzed [1] and exploited for the manipulation [2,3] and the selection of target vip receptor from a mixture in suspension [4]. Metastatic diseases, a major cause of poor prognosis, are caused by the detachment and dissemination, through the blood stream, of cancer cells from the primary tumor mass. Circulating Tumor Cells (CTC) are able to take root and continue to grow into new tissue districts very distant from the primary site, leading to a, sometimes more aggressive, new tumor development [5–7]. Moreover, the identification of the CTC and their counts in cancer patients cannot only be considered as a prognostic factor, but it can follow the trend of a certain treatment indicating possible changes and/or improvements from a simple analysis of the blood avoiding invasive biopsies [8]. The dielectrophoretic forces are generated in a non-homogeneous electric field, respectively, as positive-DEP (+DEP) (with respect to the gradient of the electric field) or negative-DEP (−DEP) cell movement under the action of this electric field is used to sort cells [9–11]. The direction of cells\' movement for a given frequency of the electric field depends on the polarizability of the cells compared with that of the extracellular medium in which they are suspended. This parameter also depends on the specific dielectric constant of the cell membrane (in turn dependent on the radius of the cells), a factor known as Clausius–Mossotti factor [12]. The exposure to a high electric field, however, induces a condition of high stress for the cells, which results in a change of the inner biochemical and biophysical properties, leading in the extreme cases either to cell death via cell lysis or apoptosis [13]. For these reasons, the cells can be manipulated for DEP only in buffer with low conductivity.
Materials and methods Both cell lines were grown in DMEM supplemented with 10% heat-inactivated FBS (Heat-Inactivated Fetal Bovine Serum, from Gibco) and antibiotics (100U/ml penicillin and 100U/ml streptomycin; Sigma-Aldrich). All cells were maintained at 37% with 95%/5% air/CO2. and are grown to 70% confluence up to 48h before being detached with trypsin (Trypsin-EDTA, Sigma-Aldrich) for MDA-MB-231 or to be re-suspended (K562). After neutralizing the trypsin with the complete medium, such type of cells was centrifuged for 5min at 1200rpm at 4°C, re-suspended in specific extracellular medium to give a final concentration of 500,000cells/ml and finally centrifuged again at the same condition. Media whit the same conductivity, osmolarity and pH were prepared mainly by varying the type of salts. Our DEP-buffer consisted of an aqueous solution of sugar with the following percentage: 9.5% sucrose (S7903, Sigma-Aldrich), 0.1mg/ml dextrose (D9559, Sigma-Aldrich), 0,1% pluronic F68 (Pluronic F68 non-ionic surfactant 100×, Gibco). This buffer has a conductivity equal to 5μS/cm and represents our elution buffer, which is then brought to the desired conductivity with solution 1M of KCl (Sigma-Aldrich) or DMEM (11965, High glucose, GIBCO). The composition of the buffers used in comparison to the total composition of the culture medium is shown in Table 1.
Results and discussion
Conclusions
Acknowledgments This research is partially supported by: PO FSE Sicilia 2007–2013, ‘Formazione di ricercatori altamente qualificati per l\'Imaging e la Sensoristica Biomedica’; CIP: 2007.IT.051.PO.003/IV/L/F/9.2/0019.