Percoll Methodology and Applications Back to Collection Amersham 18-1115-69 Biosciences Edition AC
1 18-1115-69 Edition AC Percoll Methodology and Applications
Percoll Methodology and Applications
3 Percoll Methodology and Applications
Content Introduction 7 Principles of density gradient centrifugation .8 Separation by density(lsopycnic centrifugation) Separation by size (Rate) Percoll-physical properties. .10 Particle size Sity Density. 10 pH and osmolality. .11 Behavior of the colloid. .11 How to make and use gradients of Percoll 12 Making and diluting a stock solution of Percoll 12 Diluting stock solutions to lower densities. .12 14 **4040 15 Effects of osmolality on apparent buoyant density of cells and subcellular particles. 17 Factors affecting gradient formation and shape. .18 Discontinuous gradients. .20 Continuous linear and non-linear gradients 20 Preformed self-generated gradients. 21 Gradients formed in sit 3 Maximum sample loading 22 A model experiment to standardize conditions 22 How to fractionate and analyze gradients of Percoll 23 Density determination using Density Marker Beads Using Density Marker Beads. 25 Other methods for measuring density. 25 Fractionation of gradients Cell sorting and counting 26 Protein determination and enzyme assay. Removal of Percoll after centrifugation 27 Washing 27 2 Other methods
4 Content Introduction . 7 Principles of density gradient centrifugation . 8 Separation by density (Isopycnic centrifugation) . 9 Separation by size (Rate zonal centrifugation) . 9 Percoll - physical properties . 10 Particle size composition . 10 Viscosity . 10 Density . 10 pH and osmolality . 11 Behavior of the colloid . 11 How to make and use gradients of Percoll . 12 Making and diluting a stock solution of Percoll . 12 Diluting stock solutions to lower densities . 12 The one-step procedure for diluting Percoll . 14 Diluting Percoll to a desired osmolality . 15 Effects of osmolality on apparent buoyant density of cells and subcellular particles . 17 Factors affecting gradient formation and shape. 18 Discontinuous gradients . 20 Continuous linear and non-linear gradients . 20 Preformed self-generated gradients . 21 Gradients formed in situ . 21 Maximum sample loading . 22 A model experiment to standardize conditions . 22 How to fractionate and analyze gradients of Percoll . 23 Density determination using Density Marker Beads . 23 Density Marker Beads - properties . 23 Effects of ionic strength and sucrose concentration on density of Density Marker Beads . 24 Using Density Marker Beads . 25 Other methods for measuring density . 25 Fractionation of gradients . 26 Cell sorting and counting. 26 Protein determination and enzyme assay . 26 Removal of Percoll after centrifugation . 27 Washing . 27 High speed centrifugation . 27 Other methods . 28
Practical notes Care and cleaning of equipment. Aggregates of silica particles. .29 Applications 30 Blood cells . 30 Applications-Blood Cells. Applications-Other Cell Types 38 Applications-Microorganisms. A6 Applications-Subcellular Particles. .48 Appendix 1-Summary methodology charts References. .56 Ordering Information. Culture. Products for Purification of DNA 81 Kits for cDNA Synthesis. .81
5 Practical notes . 29 Care and cleaning of equipment . 29 Storage of Percoll . 29 Sterilization of Percoll solutions . 29 Aggregates of silica particles. 29 Applications . 30 Blood cells . 30 Applications - Blood Cells. 31 Applications - Other Cell Types . 38 Applications - Microorganisms . 46 Applications - Subcellular Particles . 48 Appendix 1 - Summary methodology charts . 54 Scheme 1. Separation of cells on gradients of Percoll . 54 Scheme 2. Separation of subcellular particles on gradients of Percoll . 55 References. 56 Ordering Information. 80 Products for Cell Separation and Culture . 80 Products for Purification of RNA . 81 Products for Purification of DNA . 81 Kits for cDNA Synthesis . 81
Introduction Since its introduction in 1977,the silica colloid Percoll has become the density gradient medium of choice for thousands of researchers worldwide.Its nearly ideal physical characteristics facilitate its use in separating cells,organelles,viruses,and other subcellular particles.Percoll is especially useful as a first step to enrich for cell populations before attempting finer resolution or extraction of nucleic acids.A considerable savings of time and resources may be realized using Percoll as a first step before employing these methods. For Biological particles,the ideal gradient medium has been described as one having the following characteristics (79): .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 Percoll is exceptional among the available media in that it fulfills the above criteria,and also provides these additional advantages .It can form both continuous and discontinuous gradients. .Stability of gradients means that gradients can be premade to give reproducible results. Aag2esnpewahcoe0eayweeeataabetom .Further experiments with isolated materials are not affected by Percoll. .The success of thousands of researchers has been documented in the Percoll Reference List. This manual provides the basic methodology for making and using gradients of Percoll.In addition, n Tables in the lat s for using eppoucells,microorganisms,organelle and subcelular partce refe
7 Introduction Since its introduction in 1977, the silica colloid Percoll™ has become the density gradient medium of choice for thousands of researchers worldwide. Its nearly ideal physical characteristics facilitate its use in separating cells, organelles, viruses, and other subcellular particles. Percoll is especially useful as a first step to enrich for cell populations before attempting finer resolution or extraction of nucleic acids. A considerable savings of time and resources may be realized using Percoll as a first step before employing these methods. For Biological particles, the ideal gradient medium has been described as one having the following characteristics (79): • covers a sufficient density range for isopycnic (see p. 9 Figure 1) 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 Percoll is exceptional among the available media in that it fulfills the above criteria, and also provides these additional advantages: • It can form both continuous and discontinuous gradients. • Stability of gradients means that gradients can be premade to give reproducible results. • Analysis of gradients is simple with colored Density Marker Beads (available from Amersham Biosciences). • Further experiments with isolated materials are not affected by Percoll. • The success of thousands of researchers has been documented in the Percoll Reference List. This manual provides the basic methodology for making and using gradients of Percoll. In addition, the Application Tables in the latter part of this manual provide numerous references for using Percoll to isolate various cells, microorganisms, organelles and subcellular particles
Principles of density gradient centrifugation When a suspension of particles is centrifuged,the sedimentation rate of the particles is proportional to the force applied.The physical p ate At a fixed c ifugal fo osity,the sedi ation rate tional to the The equation for the sedimentation of a sphere in a centrifugal field is: (Pp-Pi) V=- 18m g where v=sedimentation rate d =diameter of the particle(hydrodynamically equivalent sphere) p=particle density =liquid density n =viscosity of the medium g =centrifugal force From this equation,the following relationships can be observed: .The sedimentation rate of a particle is proportional to its size The sedimentation rate is proportional to the difference between the density of the particle and that of the surrounding medium. The sedimentation rate is zero when the density of the particle is equal to the density of the surrounding medium. The sedimentation rate decreases as the viscosity of the medium increases The sedimentation rate increases as the centrifugal force increases
8 Principles of density gradient centrifugation When a suspension of particles is centrifuged, the sedimentation rate of the particles is proportional to the force applied. The physical properties of the solution will also affect the sedimentation rate. At a fixed centrifugal force and liquid viscosity, the sedimentation rate is proportional to the size of the particle and the difference between its density and the density of the surrounding medium. The equation for the sedimentation of a sphere in a centrifugal field is: where v = sedimentation rate d = diameter of the particle (hydrodynamically equivalent sphere) rp = particle density rl = liquid density h = viscosity of the medium g = centrifugal force From this equation, the following relationships can be observed: • The sedimentation rate of a particle is proportional to its size. • The sedimentation rate is proportional to the difference between the density of the particle and that of the surrounding medium. • The sedimentation rate is zero when the density of the particle is equal to the density of the surrounding medium. • The sedimentation rate decreases as the viscosity of the medium increases. • The sedimentation rate increases as the centrifugal force increases. d2 (rp - rl ) v = x g 18h
Separation by density (Isopycnic centrifugation) In this technique,the density range of the gradi- ent medium encompasses all densities of the sample particles.Each particle will sediment to an equilibrium position in the gradient where the gradient density is equal to the density of the particle(isopycnic position).Thus,in this type of on,t .o.e.e.0.o. size Figure 1 illustrates the separation(see be ypes of c r rate zonal centrifugation). When using Percoll,it is common to separate Rate zonal cent. i Isopyenic cent. Time particles isopycnically rather than on the basis of size differences(but see Figure 19,page 30,where FerClPieratrcreprcscatatioaofratczoaland both techniques are used.). y of h(a Note:When considering biological particles,it is important to remember that the osmolality of the medium can significantly alter the size and apparent buoyant density of membrane-bound rticles.A high external osmolality will caus nembra ne-b shrink while a low 1.1 lium wi cause the particle to swell. Figure 2 shov ws tha tparticles in 12 gradients of Percoll under physiological condi tions (280-320 mOs/kg H,O)have much lower apparent buoyant densities than in sucrose or Metrizamide(see also Table 1,page 18). 1d10i8021010010i00ce0s and is ed w bere u ,15■103s d by kind permiss Separation by size (Rate zonal centrifugation) In this type of separation,the size difference between particles affects the separation along with the density of the particles.As can be seen from the above equation,large particles move faster through the gradient than small particles,and the density range is chosen so that the density of the particles is greater than the density of the medium at all points during the separation(see Figure 1).The run is terminated before the separated zones reach the bottom of the tube(or their positions) 9
9 Separation by density (Isopycnic centrifugation) In this technique, the density range of the gradient medium encompasses all densities of the sample particles. Each particle will sediment to an equilibrium position in the gradient where the gradient density is equal to the density of the particle (isopycnic position). Thus, in this type of separation, the particles are separated solely on the basis of differences in density, irrespective of size. Figure 1 illustrates the two types of centrifugal separation (see below for rate zonal centrifugation). When using Percoll, it is common to separate particles isopycnically rather than on the basis of size differences (but see Figure 19, page 30, where both techniques are used.). Note: When considering biological particles, it is important to remember that the osmolality of the medium can significantly alter the size and apparent buoyant density of membrane-bound particles. A high external osmolality will cause membrane-bound particles to shrink while a low osmolality in the medium will cause the particles to swell. Figure 2 shows that particles centrifuged in gradients of Percoll under physiological conditions (280-320 mOs/kg H2O) have much lower apparent buoyant densities than in sucrose or Metrizamide (see also Table 1, page 18). Figure 1. Diagrammatic representation of rate zonal and isopycnic centrifugation. r1 = buoyant density of the less dense (blue) particles r2 = buoyant density of the more dense (red) particles (Courtesy of H. Pertoft, reproduced by kind permission.) Separation by size (Rate zonal centrifugation) In this type of separation, the size difference between particles affects the separation along with the density of the particles. As can be seen from the above equation, large particles move faster through the gradient than small particles, and the density range is chosen so that the density of the particles is greater than the density of the medium at all points during the separation (see Figure 1). The run is terminated before the separated zones reach the bottom of the tube (or their equilibrium positions). Figure 2. Approximate sedimentation rates and isopycnic banding densities of particles in a rat liver homogenate, herpes virus and human blood cells in gradients of Percoll (green) compared with sucrose gradients (blue). Svedberg units = sedimentation coefficient, 1S = 10-13 sec. (27, reproduced by kind permission of the authors and publisher). Rate zonal cent. Isopycnic cent. Time 1 2 r r r Svedberg units S 10 1.3 1.2 1.1 10 10 10 10 10 10 10 10 10 0 1 2 3 4 5 6 7 8 9 Mitochondria Mitochondria Plasma membranes Plasma membranes Lysosomes Lysosomes Microbodies Granulocytes Platelets Lymphocytes Percoll Sucrose Erythrocytes Ribosomes Ribosomes Herpes virus Herpes virus Nuclei Nuclei Buoyant density g/ml
Percoll physical properties Percoll is available from Amersham Biosciences Composition silica sol with nondialyzable polyvinylpyrrolidone(PVP)coating Density 1.130±0.005g/ml Conductivity 1.0 mS/cm Osmolality 605 can be successfully banded on gradients of Percoll,and most have buouyant densities of <1.13 g/ml in Percoll (see Figure 2). 10
10 Percoll - physical properties Percoll is available from Amersham Biosciences. Composition silica sol with nondialyzable polyvinylpyrrolidone (PVP) coating Density 1.130 + 0.005 g/ml Conductivity 1.0 mS/cm Osmolality 60S can be successfully banded on gradients of Percoll, and most have buouyant densities of <1.13 g/ml in Percoll (see Figure 2)
pH and osmolality Percoll has a pH of about 90,adjustable to pH 5.5-10.0 without any change in properties.If the pHis dropped belw5.5.ge be caused by the presenc ofdivalen cations,an effect which is s exacer Percoll has a very low osmolality (10,000 xg in an angle-head 210 the coated silica particles will begin to sediment. 1.08 This results in an uneven distribution of particles. 1.06 and thus forms a density gradient.Since Percoll is a polydisperse colloid,its component particles 1.04 will sediment at different rates.creating a verv smooth gradient.Electron microscopic analysis of 102 999 gradients by high speed centrifugation in an anglehead rotor shows that the material at the 1020 30 5 bottom of the tube is considerably enriched in iscus mm nication). ient form on by Percoll in a The etrically (i.e.less s der on the b 1.02 ln.15 MNE ting on top and mo ottom)around the xgt starting der nsity and b 63060a nes on progress ely st per with e (F ure 4 Prolonged centrifugation of Percoll at high g- forces results in all the colloid sedimenting to form a hard pellet (see "Removal of Percoll",page 27).It is important to note that if a gradient of Percoll is spun at >10,000 x g in a swinging-bucket type rotor,the colloid will rapidly sediment into a pellet and not form a suitable gradient. The colloid does not perceptibly diffuse over time,resulting in the formation of very stable gradi- ents.Therefore,both discontinuous and continuous gradients can be prepared weeks in advance, giving great reproducibility over the course of an experiment
11 pH and osmolality Percoll has a pH of about 9.0, adjustable to pH 5.5-10.0 without any change 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. Percoll has a very low osmolality (10,000 x g in an angle-head rotor, the coated silica particles will begin to sediment. This results in an uneven distribution of particles, and thus forms a density gradient. Since Percoll is a polydisperse colloid, its component particles will sediment at different rates, creating a very smooth gradient. Electron microscopic analysis of gradients by high speed centrifugation in an anglehead rotor shows that the material at the bottom of the tube is considerably enriched in larger particles (Pertoft, personal communication). The gradient forms isometrically (i.e. less dense on top and more dense on the bottom) around the starting density and becomes on average progressively steeper with time (Figure 4). Prolonged centrifugation of Percoll at high gforces results in all the colloid sedimenting to Distance from meniscus mm 0 10 1 1 2 2 3 3 4 4 20 30 40 50 60 1.02 1.04 1.06 1.08 1.10 1.12 1.14 Starting density 60 min 90 min 30 min 15 min Density g/ml form a hard pellet (see "Removal of Percoll", page 27). It is important to note that if a gradient of Percoll is spun at >10,000 x g in a swinging-bucket type rotor, the colloid will rapidly sediment into a pellet and not form a suitable gradient. 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
How to make and use gradients of Percoll Making and diluting a stock solution of Percoll In order to use Percoll to prepare a gradient,the osmolality of Percoll (undiluted)must first be adjusted with saline or cell culture medium to make Percoll isotonic with physiological salt solu- tions.Adding 9 parts (v/v)of Percoll to 1 part (v/v)of 1.5 M NaCl or 10x concentrated cell culture medium is a simple way of preparing a Stock Isotonic Percoll (SIP)solution.Final adjustment to the required osmolality can be carried out by adding salts or distilled water.Cell density depends on osmolality (see e.g.Figure 6);because of this,the osmolality of the stock solution should be checked oducibility bet mmr to Percbe e by The density of the SIP solution can be calculated from the following formula VoPo+VxP10 V=V。- (P。P) thus p;= (Pi-P10) V.+V。 where Vx volume of diluting medium(ml volume of undiluted Percoll (ml) Po = density of Percoll (1.130+0.005 g/ml:see Certificate of Analysis for exact density) P10 density of 1.5 M NaCl=1.058 g/ml (minor differences for other salts) density of 2.5 M sucrose =1.316 g/ml (minor differences for other additives) density of SIP solution produced (g/ml) Thus,for SIP in saline,=1.123 g/ml and for SIP in sucrose,=1.149 g/ml,assuming =1.130 g/ml 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. The following formula can be used to calculate the volumes required to obtain a solution of the desired density. 12
12 How to make and use gradients of Percoll Making and diluting a stock solution of Percoll In order to use Percoll to prepare a gradient, the osmolality of Percoll (undiluted) must first be adjusted with saline or cell culture medium to make Percoll isotonic with physiological salt solutions. Adding 9 parts (v/v) of Percoll to 1 part (v/v) of 1.5 M NaCl or 10x concentrated cell culture medium is a simple way of preparing a Stock Isotonic Percoll (SIP) solution. Final adjustment to the required osmolality can be carried out by adding salts or distilled water. Cell density depends on osmolality (see e.g. Figure 6); because of this, the osmolality of the stock solution should be checked routinely with an osmometer to ensure reproducibility between experiments. For subcellular particles which aggregate in the presence of salts, the Stock Isotonic Percoll (SIP) can be made by adding 9 parts (v/v) of Percoll to 1 part (v/v) of 2.5 M sucrose. The density of the SIP solution can be calculated from the following formula: 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. The following formula can be used to calculate the volumes required to obtain a solution of the desired density. where Vx = volume of diluting medium (ml) Vo = volume of undiluted Percoll (ml) ro = density of Percoll (1.130 + 0.005 g/ml; see Certificate of Analysis for exact density) r10 = density of 1.5 M NaCl = 1.058 g/ml (minor differences for other salts) density of 2.5 M sucrose = 1.316 g/ml (minor differences for other additives) ri = density of SIP solution produced (g/ml) Thus, for SIP in saline, ri = 1.123 g/ml and for SIP in sucrose, ri = 1.149 g/ml, assuming ro = 1.130 g/ml. (ro- ri ) Voro + Vxr10 Vx = Vo thus ri = (ri - r10) Vx + Vo