Equilibrium Density Gradient - Percoll

互联网2008-06-09

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Materials

Ultracentrifuge and swinging bucket rotor
Percoll
NaCl solutions with the following osmolarities:200, 300 and 400 milliosmols
Whole blood
Hemacytometer
Test tubes
Colored density marking beads

Procedure

1.For your ultracentrifuge, obtain a series of centrifuge tubes containing appropriate volumes of Percoll starting at 1.08 g/ml density as follows:
a. Percoll + colored marking beads.
b. Percoll + 200 mOsm NaCl
c. Percoll + 300 mOsm NaCl
d. Percoll + 400 mOsm NaCl

2.Use a hemacytometer to calculate the number of cells per ml of your blood sample. Carefully layer a suspension containing about 100 x 10 blood cells onto the gradients in tubes b-d.
Centrifuge the four tubes for the equivalent clearance of Beckman 30.2 rotor at 35,000 xg for 15 minutes at 20°C.

3.Remove the tubes, fractionate into 0.5 ml fractions and count the number of cells in each fraction with a hemocytometer.

4.Graph the distance from the bottom of the tube vs. the number of cells in each fraction.

5.Overlay this graph with a comparison of distance vs. density of the medium, as determined by the position of the colored beads in your control tube.

6.Compare your results to Figure 1.

Notes

Pertoft and coworkers developed a synthetic, colloidal solution of polyvinylpyrrolidone coated silica, specifically designed for sedimentation centrifugation. This material is marketed under the trade name of Percoll. Table 1 give the characteristics of this medium, compared to several other density media, namely sucrose, metrizamide and Ficoll TM .

Of particular interest is the fact that during centrifugation in a fixed angle rotor, Percoll TM will spontaneously form linear gradients, the shape of which is dependent upon rotor speed and time of centrifugation. Thus, it becomes a simple matter to establish a linear density gradient.

Figure 1 demonstrates a comparison of Percoll TM fractionation of cellular components to sucrose fractionation. This figure also presents, a standard way to comare components, by defining the relationship of density to the sedimentation coefficient for a cell or organelle. Note, that lymphocytes, granulocytes and erythrocytes have very similar sedimentation coefficients, but can be separated on the basis of density. Organelle separations are much easier to accomplish on Percoll density gradients than on sucrose gradients.

Table 1 Densities of cell structures in sucrose

<center> <table> <tbody> <tr> <td> Table 1 </td> <td> Concentration </td> <td> Density </td> <td> Viscosity </td> <td> Osmolality </td> </tr> <tr> <td> Medium </td> <td> (%w/v) </td> <td> (g/ml) </td> <td> (cP) </td> <td> (mOs/kg H 2 O) </td> </tr> <tr> <td> Sucrose </td> <td> 20 </td> <td> 1.06 </td> <td> 30 </td> <td> 700 </td> </tr> <tr> <td> Metrizamide </td> <td> 30 </td> <td> 1.16 </td> <td> 2 </td> <td> 260 </td> </tr> <tr> <td> Ficoll TM </td> <td> 30 </td> <td> 1.10 </td> <td> 49 </td> <td> 130 </td> </tr> <tr> <td> Percoll TM </td> <td> 26 </td> <td> 1.13 </td> <td> 10 </td> <td> 10 </td> </tr> </tbody> </table> </center>