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Micallef L.,University of Limoges | Battu S.,Laboratoire Of Chimie Analytique Et Bromatologie | Pinon A.,University of Limoges | Cook-Moreau J.,University of Limoges | And 3 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2010

The spontaneously immortalized human keratinocyte cell line HaCaT is widely used as a human keratinocyte model. In a previous comparative study between normal human keratinocytes (NHKs) and HaCaT, we reported that Ca2+ concentrations greater than 1 mM induced differentiation in vitro in both cell types, notably characterized by increased expression of differentiation markers keratins 1 (K1), 10 (K10) and involucrin. Surprisingly, cells had a higher proliferative activity than those cultured with low Ca2+ levels. These results raised many questions; in particular concerning the emergence of HaCaT cells subpopulation which would have different differentiation states and/or proliferation rates throughout Ca2+-induced differentiation. To isolate these subpopulations, we used sedimentation field-flow fractionation (SdFFF). Results demonstrated that the most differentiated cells (HC-F1), characterized by the highest expression of keratinocyte differentiation markers, had the lowest proliferative activity. In contrast, less differentiated cells (HC-F2) maintained a higher proliferative activity. SdFFF is a tool to sort differentiated and/or proliferating cells from a total pool previously treated with a Ca2+ concentration inducing differentiation, and can be use to prepare biological models necessary for studying HaCaT cell proliferation after Ca2+-induced differentiation treatment. © 2010 Elsevier B.V. All rights reserved.

Naves T.,University of Limoges | Battu S.,University of Limoges | Battu S.,Laboratoire Of Chimie Analytique Et Bromatologie | Jauberteau M.-O.,University of Limoges | And 4 more authors.
Analytical Chemistry | Year: 2012

The development of hypoxic areas often takes place in solid tumors and leads cells to undergo adaptive signalization like autophagy. This process is responsible for misfolded or aggregated proteins and nonfunctional organelle recycling, allowing cells to maintain their energetic status. However, it could constitute a double-edged pathway leading to both survival and cell death. So, in response to stress such as hypoxia, autophagic and apoptotic cells are often mixed. To specifically study and characterize autophagic cells and the process, we needed to develop a method able to (1) isolate autophagic subpopulation and (2) respect apoptotic and autophagic status. Sedimentation field-flow fractionation (SdFFF) was first used to monitor physical parameter changes due to the hypoxia mimetic CoCl2 in the p53 mutated SKNBE2 (c) human neuroblastoma cell line. Second, we showed that "hyperlayer" elution is able to prepare autophagic enriched populations, fraction (F3), overexpressing autophagic markers (i.e., LC3-II accumulation and punctiform organization of autophagosomes as well as cathepsin B overactivity). Conversely, the first eluted fraction exhibited apoptotic markers (caspase-3 activity and Bax increased expression). For the first time, SdFFF was employed as an analytical tool in order to discriminate apoptotic and autophagic cells, thus providing an enriched autophagic fraction consecutively to a hypoxic stress. © 2012 American Chemical Society.

Melin C.,University of Limoges | Lacroix A.,University of Limoges | Lalloue F.,University of Limoges | Pothier A.,University of Limoges | And 11 more authors.
Journal of Chromatography A | Year: 2013

SdFFF is now commonly used for cell sorting. Nevertheless, as with many other separation methods, SdFFF Hyperlayer elution leads (1) to sample dilution resulting in cell loss which could restrict further use; and (2) to a high output flow rate impacting detector sensitivity and selectivity. In order to limit these problems, we proposed modifications of the SdFFF separation channel consisting both in downscaling and the insertion of an outlet stream splitter. This last system corresponded to a strip which divides the flow rate output into two parts, one containing concentrated cells in a reduced volume and flow rate, the other containing the excess mobile phase useless for further cell manipulation, detection and characterization. For the first time we have shown that splitter implementation and downscaling respected channel flowing and resulted in Hyperlayer elution of around 95% of cells in less than 50% of input flow rate. Improved cell sorting was demonstrated by enrichment (~10 times) of cancer stem cells from WiDr cells with two times less quantity of injected cells. © 2013 Elsevier B.V.

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