Time-lapse living cells microscopy of cell line K562 for studyof CML-associated proteins dynamics - immobilization ofsuspension cultured cells

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