John L and Drescher M (2018), Xenopus laevis Oocytes Preparation for in-Cell ...

XB-ART-58395

Bio Protoc 2018 Apr 05;87:e2798. doi: 10.21769/BioProtoc.2798.

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Xenopus laevis Oocytes Preparation for in-Cell EPR Spectroscopy.

John L , Drescher M .

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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 Xenopus laevis oocytes is ideally suited for this purpose. Xenopus laevis 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 Xenopus laevis 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 Xenopus laevis oocytes by puncturing the membrane cautiously. Afterwards, three or five microinjected Xenopus laevis 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.

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Species referenced: Xenopus laevis

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References [+] :

Azarkh, Long-range distance determination in a DNA model system inside Xenopus laevis oocytes by in-cell spin-label EPR. 2011, Pubmed, Xenbase

	Figure 1. Assembly of the microinjection equipment. A. Magnified view of the control computer; B. Binocular microscope and microinjection equipment; C. Magnified view of the top of the Nanoliter Injector.
	Figure 2. Ready prepared Xenopus laevis oocytes and glass capillary. A. Magnified view of the self-made polytetrafluoroethylene holder with Xenopus laevis oocytes in MBS buffer. B. Prepared injector glass capillary clamped in the Nanoliter Injector with the self-made polytetrafluoroethylene holder.
	Figure 3. Micrographs of Xenopus laevis oocytes. A. Healthy Xenopus laevis oocytes without signs of apoptosis; B. and C. Light discolorations in the dark animal hemisphere of the Xenopus laevis oocytes as an initial sign of apoptosis are tagged by orange circles. C. Progressed apoptosis in the form of flabby membrane and cell deformation is tagged by a red ellipse. Scale bar = 1 mm.
	Figure 4. Process flow of the microinjection. A. Diagram of a Xenopus laevis oocyte; B. For the microinjection, the glass capillary penetrates the Xenopus laevis oocyte in the animal hemisphere, very close to the vegetal hemisphere, at an approximately 10° angle, tagged by a red arrow. C. Position of the glass capillary (shown in dark blue) during the injection.
	Figure 5. Prepared Q-band sample tube with three Xenopus laevis oocytes in MBS buffer immediately before shock-freezing for pulsed Q-band EPR measurements
	Figure 6. Assembly for an easy collection of the Xenopus laevis oocytes into a Q-band sample tube. A. Xenopus laevis oocytes positioned in a drop of MBS buffer on a Petri dish directly next to an elevation made out of Parafilm can be easily collected in a Q-band sample tube by means of a pipette controller. B. Magnified view of the positioning and collection of the Xenopus laevis oocytes.