Adjustment of the osmolality of Percoll for the isopycnic separation of cells and cell organelles.
The addition of 1 part of 1.5 M NaCl or 2.5 M sucrose (10 X concentrate) to 9 parts of Percoll produces a stock solution that is hypertonic (350-360 mOsm/kg H2O). Because the osmolality is a critical variable in the isopycnic separation of cells and cell organelles, the factors accountable for this hypertonicity were investigated. Percoll, a colloidal suspension of silica particles coated with polyvinylpyrrolidone, can be described as a medium composed of two distinct compartments, an aqueous phase and a solid phase. According to this model, solutes (e.g., NaCl, sucrose) should have access to the aqueous phase and add to the intrinsic osmolality of Percoll, but should be excluded from the solid phase. In order to verify this hypothesis, mathematical equations were derived and tested. It was found that the ratio of the aqueous volume to the total volume of Percoll (R value) was dependent on the ionic strength of the stock solution. With this parameter, the osmolality of Percoll stock solutions could be predicted (+/-2%) and, consequently, one could calculate the proper dilution to be used with saline, culture medium, or sucrose concentrates to obtain a truly isotonic Percoll stock solution (congruent to 290 mOsm/kg H2O). The relative importance of an accurate control of the osmolality in the preparation of density gradients made up of Percoll is also discussed.
D002458:Cell Fractionation / Q000379:methods*; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000379:methods*; D002500:Centrifugation, Isopycnic / Q000379:methods*; D003470:Culture Media; D008433:Mathematics; D009994:Osmolar Concentration; D011205:Povidone; D012822:Silicon Dioxide; D012996:Solutions; D013395:Sucrose
D003470:Culture Media; D012996:Solutions; D013395:Sucrose; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone