Percoll density gradient centrifugation Outline Principles Physical properties Storage Make and use density of Percoll Tips Protocol Instrument and operation References and applications Contrast differential velocity centrifugation For biological particles the ideal gradient medium has been described as one having the following characteristics covers a sufficient density range for isopycnic banding of all biological particles of interest possesses physiological ionic strength and pH is iso osmotic throughout the gradient has low viscosity is non toxic will not penetrate biological membranes is supplied sterile and is resterilizable will form self generated gradients by centrifugation at moderate g forces is compatible with biological materials is easily removed from purified materials does not affect assay procedures will not quench radioactive assays Principles of density gradient centrifugation Separation by density isopycnic centrifugation Separation by size rate zonal centrifugation Percoll physical properties Percoll 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 ml Conductivity 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 C Refractive Index 1 3540 0 005 at 20 C 折射率 Percoll is non toxic 1 0 1 3g ml The viscosity of Percoll is lower in saline solutions at physiological ionic Storage of Percoll Sterile and unopen 5 years When 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 disturbed If stored at 18 C gradients form upon thawing necessitating a mixing of the contents of the bottle before use Sterilization of Percoll solutions 120 C for 30 min Absence 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 Percoll Where Vx volume of diluting medium ml Vo volume of undiluted Percoll ml o density of Percoll 1 130 0 005 g ml 10 density of 1 5 M NaCl 1 058 g ml density of 2 5 M sucrose 1 316 g ml i density of SIP solution produced g ml Thus for SIP in saline i 1 123 g ml for SIP in sucrose i 1 149 g ml assuming o 1 130 g ml Adding 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 渗透活性物质的物质的量除以溶液的体积称为溶液的渗透 浓度 osmolarity 单位为mol L或mmol L How to make and use gradients of Percoll Diluting stock solutions to lower densities Solutions of Stock Isotonic Percoll SIP are diluted to lower densities simply by adding 0 15 M NaCl or normal strength cell culture medium for cell work or with 0 25 M sucrose when working with subcellular particles or viruses Tips of making and use gradients of Percoll The colloid does not perceptibly diffuse over time resulting in the formation of very stable gradients Therefore both discontinuous and continuous gradients can be prepared weeks in advance giving great reproducibility over the course of an experiment Measure the weight of the solution make sure the weight of solution are all the same 实验重复性的保证 Weight of Percoll50 55 60 65 SIP 20170914 SIP 20171014 SIP 20171114 1 Percoll was diluted 9 1 vol vol with 1 5 M NaCl 2 Put 10 ml of the Percoll solution into 15 ml Corex tubes and centrifuged at 19 240 gav for 15 min at 20 C swinging bucket rotor 3 Approximately 2 109 cells 200 OD600 were pelleted resuspended in 1 ml Tris buffer overlaid onto the preformed gradient and centrifuged at 400 gav for 60 min Isolation of quiescent and nonquiescent cells form yeast stationary phase culture Allen C The Journal of cell biology 2006 Percoll gradient purification of spores Strains were grown overnight in YPD liquid medium Cells were washed twice with ddH2O and diluted to a final cell density of OD600 0 5 Then 10 l of equal volume mixed cells were spotted on V8 medium and incubated for seven days in the dark at room temperature The entire mating patch was suspended in 60 Percoll GE Health in PBS with 0 1 Triton X100 After centrifugation at 10 000 X g for 30 mins in an SW41Ti ultracentrifuge rotor Beckman Coulter a band of spores near the bottom of the Percoll gradient was recovered with a 1 ml tuberculin syringe and transferred into an Eppendorf tube The total spore production was determined by multiplying the spore density measured by hemocytometer with the final volume Christina M Hull PLoS genetics 2015 Instrument and operation Instrument and operation Instrument and operation Instrument and operation data export References