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Exercise 8. Cell Fractionation II: Isolation and Visualization of Liver Cell Nuclei Using Density Gradient Centrifugation and Fluorescence Microscopy

 


A. Introduction

Image source: Wikipedia

As first discussed in exercise #5, the study of the structure, function and biochemistry of cellular compartments and organelles often requires the isolation of these cellular components, a process called cell fractionation. In exercise #5, basic cell fraction techniques were used to produce relatively pure fractions of mammalian red blood cell plasma membrane. In that exercise, simple osmotic stress was used to lyse red blood cells, and differential centrifugation was used to isolate the plasma membrane.

For obvious reasons, mature mammalian red blood cells, while the perfect choice for the isolation and study of plasma membrane, cannot be used for the isolation of nuclei or cytoplasmic organelles. Fractionation of nuclei and cytoplasmic organelles requires other tissues and often times other fractionation techniques. Liver has been one of the tissues of choice for the development of cell fractionation techniques for nuclei and cytoplasmic organelles. Liver tissue is a soft tissue with relatively sparse amounts of connective tissue, making it an ideal tissue for thes structures. Thus, liver cell fractionation techniques are widely used and are well documented.

Unlike the techniques used for mammalian red blood cell plasma membrane fractionation, the established fractionation techniques used for liver cells require mechanical tissue homogenization and a type of centrifugation known as density gradient centrifugation. The specific objective of today's experiment is to create relatively pure fractions of nuclei from rat liver using tissue homogenization and density gradient centrifugation. Once isolated, the nuclei will be visualized using fluorescence microscopy.