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 <25 mOs/kg H2O Viscosity 10±5cPat20C pH 9.0±0.5at20C Refractive Index 1.3540±0.005at20 Percoll is non-toxic Particle size composition The physical properties of Percoll have been extensively studied by Laurent et al.(45,46,47). Electron microscopic examination(Figure 3) shows the silica to be in the form of a nea 0f21-22nm n particle dia 8 29 nd water, 100 nm for the mean particle diameter, Figure 3.E indicating a layer of hydration on the particles which is more pronounced at low ionic strength. Chromatography of Percoll on Sepharose4B(22)has demonstrated the presence of only 1-2% free PVP.Inclusion of PEG in the eluant did not result in any loss of PVP from the silica,indicating that the PVP is firmly bound.Calculations based on the nitrogen content of the colloid indicate that the pVP coating is a monomolecular laver. Viscosity The viscosity of Percoll is a function of the ionic strength,and is lower in saline solutions at physi- ological ionic strength (e.g.0.15 M NaCl)than in water or in 0.25 M sucrose (22). This has the effect of making gradient formation in 0.15 M NaCl much faster than in 0.25 M sucrose when solutions are centrifuged under identical conditions(page 18).Under working conditions,the viscosity of Percoll solutions is 1-15 cP,facilitating extremely rapid banding of particles in gradients of Percoll. Density Percoll is supplied as a23%(w/w)colloidal solution in water having a density of 1.1300.005 g/ml Gradients ranging from 1.0-1.3 g/ml are achievable by centrifugation as described elsewhere in this booklet.All biological particles having sedimentation coefficient values of >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 <25 mOs/kg H2O Viscosity 10 + 5 cP at 20 ºC pH 9.0 + 0.5 at 20 ºC Refractive Index 1.3540 + 0.005 at 20 ºC Percoll is non-toxic Particle size composition The physical properties of Percoll have been extensively studied by Laurent et al. (45, 46, 47). Electron microscopic examination (Figure 3) shows the silica to be in the form of a polydisperse colloid composed of particles from 15 to 30 nm in size, with a mean particle diameter of 21-22 nm. Hydrodynamic measurements (viscometry and sedimentation) give values of 29- 30 nm and 35 nm in 0.15 M NaCl and water, respectively, for the mean particle diameter, indicating a layer of hydration on the particles which is more pronounced at low ionic strength. Figure 3. Electron microscopy of Percoll particles. Negative contrast with 1% uranyl acetate at pH 4.6 (21, reproduced by kind permission of the authors and publisher). Chromatography of Percoll on Sepharose™ 4B (22) has demonstrated the presence of only 1-2% free PVP. Inclusion of PEG in the eluant did not result in any loss of PVP from the silica, indicating that the PVP is firmly bound. Calculations based on the nitrogen content of the colloid indicate that the PVP coating is a monomolecular layer. Viscosity The viscosity of Percoll is a function of the ionic strength, and is lower in saline solutions at physiological ionic strength (e.g. 0.15 M NaCl) than in water or in 0.25 M sucrose (22). This has the effect of making gradient formation in 0.15 M NaCl much faster than in 0.25 M sucrose when solutions are centrifuged under identical conditions (page 18). Under working conditions, the viscosity of Percoll solutions is 1-15 cP, facilitating extremely rapid banding of particles in gradients of Percoll. Density Percoll is supplied as a 23% (w/w) colloidal solution in water having a density of 1.130 + 0.005 g/ml. Gradients ranging from 1.0-1.3 g/ml are achievable by centrifugation as described elsewhere in this booklet. All biological particles having sedimentation coefficient values of >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 (<25 mOs/kg HO)and can therefore form a density gradient thout pro ucing density gradi This ma kesit possible e to work wit smotic and adjusted to physiological cond ons through out.This is very importan r obtaining preparations of cells having extremely high viabilities(23),and intact morphology (31).Due to this fact,gradients of Percoll also provide an opportunity to observe the effect of osmolality on the apparent buoyant density of cells and subcellular particles(see page 17 and ref.27). Behavior of the colloid Percoll particles have an inner core of silica which is very dense (p=2.2 g/ml)and an 1.141 ated particle size of 29-30 nm in 0.15 M NaCl 112 wyater (46)Thu when a solutio of Percoll (in 0.15 M saline or 0.25 Ms ose)is centrifug at>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 (<25 mOs/kg H2O) and can therefore form a density gradient without producing any significant osmolality gradient itself. This makes it possible to work with density gradients which are iso-osmotic and adjusted to physiological conditions throughout. This is very important for obtaining preparations of cells having extremely high viabilities (23), and intact morphology (31). Due to this fact, gradients of Percoll also provide an opportunity to observe the effect of osmolality on the apparent buoyant density of cells and subcellular particles (see page 17 and ref. 27). Behavior of the colloid Figure 4. Isometric gradient formation by Percoll in an anglehead rotor, 8 x 14 ml (MSE Superspeed centrifuge) starting density 1.07 g/ml in 0.15 M NaCl. Running conditions: 20,000 x g for 15, 30, 60 and 90 minutes. Gradient density was monitored by means of colored Density Marker Beads. See Figure 12, page 23 (Work from Amersham Biosciences, Uppsala, Sweden). Percoll particles have an inner core of silica which is very dense (r = 2.2 g/ml) and an average hydrated particle size of 29–30 nm in 0.15 M NaCl and 35 nm in water (46). Thus, when a solution of Percoll (in 0.15 M saline or 0.25 M sucrose) is centrifuged at >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