Percoll-密度梯度离心教程演示教学.pptxVIP

Percoll density gradient centrifugationOutlinePrinciplesPhysical propertiesStorageMake and use density of PercollTipsProtocolInstrument and operationReferences and applicationsContrast(differential velocity centrifugation)Principles of density gradient centrifugation?Separation by density(isopycnic centrifugation)Separation by size (rate zonal centrifugation)Percoll – physical propertiesPercoll is available from GE Healthcare.Composition silica sol with nondialyzable polyvinylpyrrolidone (PVP) coating（由聚乙烯吡咯烷酮包裹的硅胶颗粒）Density 1.130 ± 0.005 g/mlConductivity 1.0 mS/cm（电导率）Osmolality 25 mOsm/kg H2O（渗透压）Viscosity 10 ± 5 cP at 20°C（粘度）pH 9.0 ± 0.5 at 20°CRefractive Index 1.3540 ± 0.005 at 20°C（折射率）Percoll is non-toxic1.0~1.3g/ml The viscosity of Percoll is lower in saline solutions atphysiological ionicStorage of PercollSterile and unopen----5 yearsWhen opened, Percoll should be stored below +8℃If Percoll opened under non-sterile conditions, it can be frozen for up to 6minths at -18℃Preformed gradients can be stored for weeks without a change in gradient shape, provided that the gradient is sterile and is not physically disturbedIf stored at -18°C, gradients form upon thawing, necessitating a mixing of the contents of the bottle before use.Sterilization of Percoll solutions120°C for 30 minAbsence of salts or sucrose.Minimum contact with air (narrow-necked bottle)If Percoll form particles, these particles may be removed by low speed centrifugation. If any significant evaporation occurs during autoclaving, the volume should be replenished with sterile water so that the density is not affected.How to make and use gradients of Percoll—Making and diluting a stock solution of PercollAdding 9 parts (v/v) of Percoll to 1 part (v/v) of 1.5 M NaCl or 10× concentrated cell culture medium is a simple way of preparing a Stock Isotonic Percoll (SIP) solution.Where Vx = volume of diluting medium (ml) Vo = volume of undiluted Percoll (ml) ρo = density of Percoll (1.130 + 0.005 g/