Cell fractionation is the process of producing pure fractions of cell components. The process involves two basic steps: disruption of the tissue and lysis of the cells, followed by centrifugation.
Click here for an overview of the cell fractionation process.
Tissue Disruption and Lysis.
The first step in cell fractionation is tissue disruption and cell lysis. The objective is to disaggregate the cells and break them open with minimum damage to the cellular fraction of interest (i.e., don't use a hammer). Tissues can be broken up and cells lysed in a number of ways. The three basic methods of breaking up the tissues and cells are 1) homogenization, 2) sonication, and 3) osmotic lysis. The particular method one chooses depends on the tissue, the cell type, and the particular cell fraction of interest. Most animal and plant tissues must be homogenized. Homogenization involves the use of a mechanical homogenizer, like a blender or a mortor and pestle, to break the tissue apart and lyse the cells. Sonication involves the use of ultrasound to disrupt the cells. Sonication is often used when prokarytic cells are to be lysed. Osmotic lysis is often the method of choice when dealing with cells that are vulnerable to osmotic stresses. Red blood cells are a perfect example of a cell that can easily be lysed through osmotic stress.
Lysing Mammalian Red Blood Cells
Mammalian red blood cells are very sensitive to the tonicity of the surrounding fluid. In vivo, mammalian red cells are bathed in isotonic plasma, in which case there is no net osmosis and the cell neither shrinks or swells. We learned in last week's lab that when placed in a sufficiently strong hypotonic fluid (e.g. NaCl solution << 0.85% NaCl), there is an uncontrolled net inward osmosis, resulting in the continuous uncontrolled swelling of the cells until the cells lyse, or hemolyze. Thus, to lyse mammalian red blood cells as part of a cell fractionation process, one can simply add cells to a hypotonic fluid.
One cannot use any hypotonic fluid however. The fluid must contain an adequate concentration of physiological salts to buffer the solution at a physiological pH so the the plasma membrane remains functional. Therefore, the fluid can only be mildly hypotonic. We saw last week that a mildly hypotonic fluid will likely not hemolyze 100% of the cells, so we need to help the process along by shaking the cells.
The second step in the cell fractionation process is centrifugation. Most of the cellular components in a cell lysate will eventually, given time, settle to the bottom of a tube. To accelerate this process, the lysate can be subjected to centrifugation. In centrifugation, the lysate is rotated at a certain speed (expressed as rotations per minute (RPM)). This rotation imposes a force on the particles perpendicular to the axis of rotation. The force is called a relative centrifugal force (RCF), expressed as a multiple of the force of Earth's gravitational force (x g). For example, an RCF of 1000 x g is a force 1000 times greater than Earth's gravitational force. When a particle is subjected to centrifugal force, it will migrate away from the axis of rotation at a rate dependent on the particle's size and density.
That part of the centrifuge that holds the centrifugation tubes is called the centrifuge rotor. Centrifuges are designed so that a number of different rotors can be used by the instrument. There are three type of centrifuge rotors: fixed angle rotors, swinging bucket rotors, and vertical rotors.
Fixed-angle and swinging-bucket rotors are the most commonly used. In a fixed-angle rotor, the centrifuge tubes are spun at a fixed angle, which is usually approximately 35 degress. Fixed angle rotors are most commonly used for pelleting cells and subcellular components. With swinging-bucket rotors, the tubes are free to swingout perpendicular to the axis of rotation as the rotor rotates. This rotor is particularly useful in density-gradient centrifugation schemes.
Differential centrifugation is one of two major types of centrifugation schemes. Differential centrifugation is the sequential centrifugation of a cell lysate at progressively increasing centrifugation force, isolating cellular components of decreasing size and density. The separation of the cellular components is based solely on their sedimentation rate through the centrifugation medium, which, in turn, is dependent on the size and shape of the cellular components.
In differential centrifugation, each centrifugation step results in the production of a pellet, usually containing a mixture of cellular components of the same size and/or density. The fluid resting above the pellet, the supernatant, can be removed and subjected to additional centrifugations to generate pellets containing other cellular components of lesser size and / or density.
Fractionation Methods to be Employed in This Week's lab
In this week's lab, we will use osmotic lysis combined with rigorous shaking to lyse mammalian red blood cells. This will be followed by simple differential centrifugation to form a pellet of red blood cell plasma membranes. The membrane pellet will need to be washed and re-centrifuged several times to was away as much of the cell cytoplasm as possible. The relatively pure membranes will then be frozen for future use.
Cell Biology OLM | Authored by Stephen Gallik, Ph. D. | URL: cellbiologyolm.stevegallik.org | Copyright © 2011 Stephen Gallik, Ph. D.