Time-lapse living cells microscopy of cell line K562 for study of CML-associated proteins dynamics - immobilization of suspension cultured cells
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| Year of publication | 2009 |
| Type | Conference abstract |
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| Description | Chronic myelogenous leukemia (CML) is characterized by genetic abnormality, the Philadelphia chromosome carrying BCR/ABL gene. This gene generates an aberrant kinase p210 (or less frequent type p190 or p230) whose unregulated activity mainly participates in the hematopoietic stem cells malignant transformation (Barnes and Melo, 2002). To fully understand the leukemogenesis it is important to describe not only p210 kinase function, but also the roles of native ABL1 and BCR kinases in the cell. We focus on localization and dynamics of these proteins in living leukemic cells that can bring new insights into the protein function. Time-lapse microscopy of living cells is a method allowing us to study proteins in their natural environmental conditions in real time. Thanks to fluorescent proteins it is possible to examine protein localization and moreover protein dynamics or protein-protein interactions in living cells (Rustom et al., 2000). Adherent cells are mainly used for this type of methods, since these cells are not mobile or subject to gravity. However, we work with suspension cells such as K562 or CD34+ hematopoietic stem cells in our CML study. Living-cell microscopy of non-adherent cells is complicated and seldom done, because it is necessary to immobilize suspension cultured cells to coverglass to protect them from the microscope stage movements, objective focus, and media flow caused by additional pipetting. It is also crucial to maintain the viability of the cells during such experiment and not to alter it by immobilization techniques. There are different ways to immobilize cells in 2D or 3D mode. For 2D mode it is possible to use surfaces coated with poly-L-lysine, chemically etched glass, fibronectin, collagen, and so forth. Unfortunately these surfaces although change shape and viability of suspension cells. Thus they are not very suitable for living-cells experiments. Therefore, we tested the immobilized culture of K562 cells on poly-L-lysin coated LabTec chamber. We compared results with novel method using oleyl poly (ethylene glycol) ether (SUNBRIGHT OE-020CS) modified LabTec chamber, suitable for studying suspension cells with maximal viability, unaffected cell growth, and maintained cell shape (Kato et al., 2003). Using BAM, K-562 cells were well fixed without any movements in X and Y axis. They preserved round shape and proliferated well. Limitation of this method lies in weak resistance of bond to liquid flow. We also noticed cell bottom alteration after longer incubation periods. |
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