If stored at -20°C, gradients form upon thawing, necessitating a mixing of the bottle before use. Performed gradients can be stored for weeks without a change in gradient shape, provided that the gradient remains unfrozen.
⋅ Percoll® can be buffered within the pH range 5.5 to 10.0 without any changes in properties. If the pH is dropped below 5.5, gelling may occur. Gelling can also be caused by the presence of divalent cations, an effect which is exacerbated by elevated temperatures.
⋅ Undiluted Percoll® can be resterilized by autoclaving for 30 min at 120°C.Preparation of gradient material
Percoll® PLUS (sc-296039
)/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®diluted with sucrose (0.25 M final concentration).
The low osmolality of Percoll® 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® 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® 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®
Percoll® 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® prior to centrifugation and will band isopycnically, as the gradient is formed in situ. Although Percoll® 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®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® by dilution with physiological saline and centrifugation to collect the cells.
• Subcellular particles can be separated from Percoll® by the procedure described above. The size of the particles will determine the centrifugal force required to separate the particles from Percoll®.
• Gel filtration or ion exchange chromatography can also be used to separate biological material from Percoll®.Aggregates of silica particles
It is an inherent tendency of all silica colloids to form aggregates on prolonged storage. These aggregates may be observed in some batches of Percoll®, either as a slight sediment at the bottom of the tube or as a faint white band with a density of 1.04 to 1.05 g/ml. This band may form during gradient formation in the centrifuge or during low speed centrifugation of a performed gradient. The aggregated silica does not interfere with the separation of biological particles and almost all cells and organelles have buoyant densities of greater than 1.05 g/ml in PPercoll® PLUS For the majority of cell, virus, and organelle separations, any silica aggregates banded from the gradient material (see above) may be ignored.
For specific experiments, it may be desirable to remove aggregates; this may be achieved by filtration of Percoll® through a depth filter prior to centrifugation.
Aggregation is not a problem in Percoll® PLUS (sc-296039
) due to the silane coating.