用法 :
• Performed gradients can be stored for weeks without change in gradient shape, provided that the gradient remains unfrozen.
• Percoll® PLUS can be buffered within the pH range 5.5 to 10 without change in properties.
• Concentrated or diluted Percoll® PLUS can be resterilized by autoclaving for 30 min at 120°C.
Preparation of gradient material
Percoll® PLUS/Percoll® are best used in balanced salt solutions, physiological saline, or 0.25 M sucrose. Cells can be separated on gradients in balanced salt solutions. Subcellular particles, however, tend to aggregate in the presence of salts and it is recommended that the separation of such particles be carried out in Percoll® PLUS diluted with sucrose (0.25 M final concentration).
The low osmolality of Percoll® PLUS permits this parameter to be controlled by the user without significant interference from the density medium itself. The addition of 9 parts (v/v) of Percoll® PLUS to 1 part (v/v) of either 1.5 M NaCl, 10x concentrated
cell culture medium, or 2.5 M sucrose will result in a solution adjusted to about 340 mOsm/kg H2O. Solutions of different osmotic pressure can be produced by adjusting the relative volumes of Percoll® PLUS and salt or sucrose solution. The final adjustment to the required osmolality can be carried out by the addition of salts or distilled water. When precise osmotic pressures are required, it is recommended that the osmolality of the solutions be measured in an osmometer.
Centrifugation with Percoll® PLUS
Percoll® PLUS will form self-generated gradients by
centrifugation at approximately 10,000 gav (in 0.15 M saline) or 25,000 gav (in 0.25 M sucrose) in fixed-angle rotor heads after 15 minutes. Cells or subcellular particles can be mixed with Percoll® PLUS prior to centrifugation and will band isopycnically, as the gradient is formed in situ. Although Percoll® PLUS is best used in angle-head rotors, banding of cells on pre-formed (continuous or discontinuous) gradients may be carried out at 400 gav for 20 to 30 minutes in swing-out rotors.
Removal of Percoll® PLUS after centrifugation
To remove the gradient medium from the biological material, perform one of the procedures outlined below.
• Cells can be recovered free from particles of Percoll® PLUS by dilution with physiological saline and centrifugation to collect the cells.
• Subcellular particles can be separated from Percoll® PLUS by the procedure described above. The size of the particles will determine the centrifugal force required to separate the particles from Percoll® PLUS.
• Gel filtration or ion exchange chromatography can also be used to separate biological material from Percoll® PLUS.
