Oocytes Preparation for in-Cell EPR Spectroscopy.

One of the most exciting perspectives for studying bio-macromolecules comes from the emerging field of in-cell spectroscopy, which enables to determine the structure and dynamics of bio-macromolecules in the cell. In-cell electron paramagnetic resonance (EPR) spectroscopy in combination with micro-injection of bio-macromolecules into oocytes is ideally suited for this purpose. oocytes are a commonly used eukaryotic cell model in different fields of biology, such as cell- and development-biology. For in-cell EPR, the bio-macromolecules of interest are microinjected into the oocytes upon site-directed spin labeling. The sample solution is filled into a thin glass capillary by means of Nanoliter Injector and after that microinjected into the dark animal part of the oocytes by puncturing the membrane cautiously. Afterwards, three or five microinjected oocytes, depending on the kind of the final in-cell EPR experiment, are loaded into a Q-band EPR sample tube followed by optional shock-freezing (for experiment in frozen solution) and measurement (either at cryogenic or physiological temperatures) after the desired incubation time. The incubation time is limited due to cytotoxic effects of the microinjected samples and the stability of the paramagnetic spin label in the reducing cellular environment. Both aspects are quantified by monitoring cell morphology and reduction kinetics.