Table 1.Changes in buoyant density of lysosomes after incubation in serum albumin. of ym 8.5 284 1.045 28 1.05 10 85 310 20 85 374 1110 8.5 503 1.148 40 8.5 800 1.177 A ly mal fraction from rat hep oll/0.25 M su fth The Factors affecting gradient formation and shape Although the hydrated volume of Percoll particles is smaller in the presence of 0.15 M NaCl than in Percoll/0.25 M sucrose,the sedimentation rate of the particles is faster due to the lower viscosity of Percoll in saline.Thus,when Percoll is made iso- osmotic with a final concentration of 0.15 M saline or a tissue culture medium of equivalent ionic strength,it will form a self-ger ated gradient about 7.3 time han the alent P coll soluti made 0sm oncentration of 0.25 M sucrose n and ti ime are interr elated in that it is the tota -forc x (ti me)which determines the shape of th 14 nt.A approximately saline and about 25,000 x g for Percoll in 0.25 M sucrose in order to self-generate gradients in anglehead rotors.Rotor geometry has a marked effect on gradient shape under given conditions as 100 110 120 shown in Figure 8.As the angle approaches vertical, the path-length for formation of the gradient 8.The angle c becomes shorter and the gradient forms more s 9 and 10 dem rate that 0hc3000U of Percoll also has rker bea some effect luced by kind permission of the authors and on the shape of th e gradient formed be tak n,how may be formed under high speed centrifugation conditions does nor contaminate the gradient during fractionation. 18
18 Factors affecting gradient formation and shape Figure 8. The effect of rotor angle on gradient development using Percoll. Starting density was 1.065 g/ml in 0.15 M NaCl. Running conditions: 30,000 x g for 14 minutes. Colored lines refer to the positions of the colored density marker beads. (45, reproduced by kind permission of the authors and publisher.) Table 1. Changes in buoyant density of lysosomes after incubation in serum albumin. Incubation medium Osmolality of Average density Albumin % Sucrose % medium (mOm/l) of lysosomes (g/ml) - 8.5 284 1.045 2.5 8.5 288 1.058 5 8.5 292 1.074 7.5 8.5 300 1.078 10 8.5 310 1.091 20 8.5 374 1.110 30 8.5 503 1.148 40 8.5 800 1.177 A lysosomal fraction from rat hepatocytes was recovered from a Percoll/0.25 M sucrose gradient at a density of 1.0 - 1.05 g/ml and incubated in the media described in the table for 1 hour at 37 ºC. The buoyant density was then redetermined in a gradient of Percoll/0.25 M sucrose (27, reproduced by kind permission of the authors and publisher). 8 6 4 2 6 4 2 6 4 2 40º 23.5º 14º Density g/ml Distance from the meniscus cm 1.00 1.10 1.20 Although the hydrated volume of Percoll particles is smaller in the presence of 0.15 M NaCl than in Percoll/0.25 M sucrose, the sedimentation rate of the particles is faster due to the lower viscosity of Percoll in saline. Thus, when Percoll is made isoosmotic with a final concentration of 0.15 M saline or a tissue culture medium of equivalent ionic strength, it will form a self-generated gradient about 2-3 times faster than the equivalent Percoll solution made iso-osmotic with a final concentration of 0.25 M sucrose. Centrifugation and time are interrelated in that it is the total (g-force) x (time) which determines the shape of the gradient. A minimum of approximately 10,000 x g should be used for Percoll in 0.15 M saline and about 25,000 x g for Percoll in 0.25 M sucrose in order to self-generate gradients in anglehead rotors. Rotor geometry has a marked effect on gradient shape under given conditions as shown in Figure 8. As the angle approaches vertical, the path-length for formation of the gradient becomes shorter and the gradient forms more rapidly. Figures 9 and 10 demonstrate that the initial concentration of Percoll also has some effect on the shape of the gradient formed. Centrifugation in vertical rotors will form gradients of Percoll very rapidly. Care must be taken, however, to ensure that the compacted pellet of Percoll which may be formed under high speed centrifugation conditions does nor contaminate the gradient during fractionation
The use of swinging bucket rotors for self-generation of gradients is not recommended,due to the long path length and unequal g-force along the tube. L12 er Jenkins et al.(pe tion 90% and d ref.87) 110 80% e types o tors for subcellua fractic nation of liver organelles 1.08 709 60 Zonal rotors 1.06. 50% have the same characteristics as those generated 1.04 40 in angle-head rotors.Because of their large sample volumes,it is recommended that the 20% separation conditions in a nonzonal rotor be empirically determined prior to scale-up in a zonal rotor.Zonal rotors have been used in the nsity Marker Beads to s large scale purification of viruses(21)and for subfractionation of lysosomes(24). 23 .000 When starting work with self-generated gradients with c colorcdoconducta Sweden.) it is advisable 22 standard cu 114 characteristic of the e angle 1.12 head rotor to be used for subsequent experiments. 1.08 1.06 1.04 1.02 re 10.Use of lored De 23"angle 000 x g.15 minutes oscic 9
19 Figure 10. Use of colored Density Marker Beads to show gradient shapes. Dilutions of Percoll as in Figure 9, running conditions: 23º angle-head rotor, 60,000 x g, 15 minutes. Steeper gradients were formed by the greater g-force. (Work from Amersham Biosciences, Uppsala, Sweden.) Figure 9. Use of colored Density Marker Beads to show gradient shape. Gradients formed from solutions of Percoll varying from 90% to 20% of stock isotonic Percoll in 0.15 M NaCl. Running conditions 23º angle-head rotor 30,000 x g, 15 minutes. (Work from Amersham Biosciences, Uppsala, Sweden.) 60 50 40 30 20 10 20% 30% 40% 50% 80% 60% 70% 90% Density g/ml Band position from bottom of tube mm 1.02 1.04 1.06 1.08 1.12 1.14 1.10 Density g/ml 1.02 1.04 1.06 1.08 1.12 1.14 1.10 60 50 40 30 20 10 Band position from bottom of tube mm 20% 30% 40% 50% 80% 60% 70% 90% The use of swinging bucket rotors for self-generation of gradients is not recommended, due to the long path length and unequal g-force along the tube. However Jenkins et al. (personal communication and ref. 87) report some advantages in using these types of rotors for subcellular fractionation of liver organelles. Zonal rotors can be used to form gradients of Percoll in situ. Gradients formed in zonal rotors have the same characteristics as those generated in angle-head rotors. Because of their large sample volumes, it is recommended that the separation conditions in a nonzonal rotor be empirically determined prior to scale-up in a zonal rotor. Zonal rotors have been used in the large scale purification of viruses (21) and for subfractionation of lysosomes (24). When starting work with self-generated gradients, it is advisable to conduct a model experiment with colored Density Marker Beads (see page 22) to produce a series of standard curves under known conditions which are characteristic of the anglehead rotor to be used for subsequent experiments
Discontinuous(step)gradients Discontinuous gradients offer great flexibility and Banded cells ease of use.Often,only a cushion of Percoll or a single step is all that's required to achieve excellent enrichment or resolution of a target cell type.For example,most blood cells can be enriched using discontinuous gradients(66,69)(see also Figure 11). 390×g 1.06f 30 min 1.068 To form a discontinuous gradient.SIP is diluted 1.070 to a series of different densities as described on page 12.The solutions of different density are 1.080 carefully layered in order of density PBMC in tin the Percoll solution 11.Sepa keep the tip of the instrument against the wall of the tube just above the surface of the liquid to avoid a splash"and mixing at the interface.Formatior of a sharp band of cells at a interface will occur only if there is a sharp change in density. Centrifugation is performed using relatively gentle condition,such as 400 x g for 15-20 minutes in a will not affect the g adient in any Continuous linear and non-linear gradients Continuous gradients are characterized by a smooth change in density from the top to the bottom of the tube.Instead of the obvious interfaces present in the discontinuous gradient,a continuous gradient can be thought of as having an infinitive number of interfaces.Therefore,isopycnic banding of cells occurs at the precise density of the cell. solutions of known density at the limits of the rang red nd ther ces G (e.g.Am GM-1).A linear gradient spanning the range between the limits of the two starting solutions is formed Asingle-channel peristaltic pump(e.Amersham Biosciences Peristaltic Pump P-1)in combination with a grad ient mixer can be used to generate linear,convex,and concave gradients,depending upon the relative diameters of the tubing used.A very narrow range of densities from top to bottom of the gradient can be formed to effect a maximum resolution of viable cells.Heavier cells usually pellet,while non-viable cells are found at the top of the gradient.For example,erythrocytes will pellet if the density at the bottom of the gradient does not exceed 1.08 g/ml.Density Marker Beads can be used as an external marker in a tube containing an identical gradient to that in the sample tube. 20
20 Discontinuous (step) gradients Discontinuous gradients offer great flexibility and ease of use. Often, only a cushion of Percoll or a single step is all that's required to achieve excellent enrichment or resolution of a target cell type. For example, most blood cells can be enriched using discontinuous gradients (66,69) (see also Figure 11). To form a discontinuous gradient, SIP is diluted to a series of different densities as described on page 12. The solutions of different density are then carefully layered in order of density one on top of another, starting with the most dense at the bottom of the tube. This is most conveniently done using a pipette or a syringe fitted with a wide-bore needle. It is important to keep the tip of the instrument against the wall of the tube just above the surface of the liquid to avoid a "splash" and mixing at the interface. Formation of a sharp band of cells at a interface will occur only if there is a sharp change in density. Centrifugation is performed using relatively gentle condition, such as 400 x g for 15-20 minutes in a bench-top centrifuge. These gentle conditions result in the isopycnic banding of cells at the relevant interfaces. The low-g conditions and short run time will not cause sedimentation of the Percoll and will not affect the gradient in any way. Continuous linear and non-linear gradients Continuous gradients are characterized by a smooth change in density from the top to the bottom of the tube. Instead of the obvious interfaces present in the discontinuous gradient, a continuous gradient can be thought of as having an infinitive number of interfaces. Therefore, isopycnic banding of cells occurs at the precise density of the cell. To form such a gradient, SIP is first diluted to produce two solutions of known density at the limits of the range required, and then mixed using a dual-chamber gradient maker (e.g. Amersham Biosciences Gradient Mixer GM-1). A linear gradient spanning the range between the limits of the two starting solutions is formed. A single-channel peristaltic pump (e.g. Amersham Biosciences Peristaltic Pump P-1) in combination with a gradient mixer can be used to generate linear, convex, and concave gradients, depending upon the relative diameters of the tubing used. A very narrow range of densities from top to bottom of the gradient can be formed to effect a maximum resolution of viable cells. Heavier cells usually pellet, while non-viable cells are found at the top of the gradient. For example, erythrocytes will pellet if the density at the bottom of the gradient does not exceed 1.08 g/ml. Density Marker Beads can be used as an external marker in a tube containing an identical gradient to that in the sample tube. PBMC in Percoll solution Banded cells 30 min 390 x g 1 2 3 4 5 6 7 1.070 1.068 1.066 1.064 1.062 1.004 Fraction number Density of Percoll solution g/ml 1.080 Figure 11. Separation of lymphocytes and monocytes by discontinuous density centrifugation in Percoll. 1.5-2.0 x 107 PBMC (peripheral blood mononuclear cells) isolated on Ficoll Paque were mixed in 11.25 ml of Percoll in Hanks BSS containing 1% HEPES buffer (density = 1.080 g/ml) and underlayered below the steps shown in the figure (69, reproduced by kind permission of the authors and publisher)
The centrifugation conditions necessary to achieve a separation are the same as those for the discontinuous gradients.Examples of separations performed on continuous gradients include the purification of Leydig cells(31),lactotrophs(19),bone marrow cells(52),intestinal epithelial cells (18,ma algae (28.60)and chlor oplasts(49,58,76,88,109 Preformed self-generated gradients Preforming a gradient by centrifugation can be a convenient alternative to using a gradient maker or pump.As described earlier,Percoll will sediment when subjected to significant g-forces (i.e.>10,000 x g). When preforming a gradient,SIP is diluted to a density that lies in the middle of the range in which maximum resolution is required.Two centrifuge tubes are filled with gradient material -one for the eriment and one conta ing Density Marker Beads.This second tube serves both as a counter- thod fo onit dient.The tubes entrifuged in angle-head g.30.000 xg for 15m orine es). and th e gra nd the starting density (Figure 4. "at omp ang required resolution o target cells. This be confirmed observing the shape of the grac ent in th ube containing the Density Mar ker Bea .The gradient becom s progressively steeper with time.It has been shown that the shape of the gradient is approximately linear related to the total g-force and time of the centrifugation (22). After forming the gradient,isopycnic banding of cells can be accomplished by low-speed centrifugation for 15-20 minutes at 400 x g.If an estimate of cell density is required,a volume equal to that of the cell suspension is layered on top of the tube containing the Density Marker Beads,.This serves as both a way to estimate cell density and as a counter-balance. Gradients formed in situ The sedimentation coefficients of subcellular particles and viruses are usually too low to allow banding on preformed gradients at low g-forces.Therefore,it is often convenient to mix the suspension of biological particles with Percoll and to band the particles on a gradient formed in situ. Gradients of percoll formed by centrifugation are metastable-ie.they will change continuously during high speed centrifugation.The rate of sedimentation of the colloid is slow enough to allow the banding of small viruses and cell organelles with "S"values >605 as the gradient is formed in situ. A common method for forming gradients in situ is to prepare a SIp,using 9 parts of Percoll to 1 part of 2.5 M sucrose.The SIP is ther n diluted to the desired density ing 0.25 M sucrose.(Although make in situ gradients,cell cult media can also be used).Wher ly with g rial,the effect on the rall der of the Percoll from th a on page 13.Premixin gradi n en en it is desir able ty of the par ever,it may be bette to lay the experimenta ularly in cases where it is The iwill remain in the buffer laver above the gradient and subcellular pa separate in the Percoll gradient in situ. Centrifugation must be carried out in an angle-head rotor.A balance tube containing Densitv Marker Beads in place of experimental sample is used to monitor the gradient.An appropriate model experiment similar to the one described on page 22,should be carried out first to establish the gradient formation characteristics of the rotor to be used
21 The centrifugation conditions necessary to achieve a separation are the same as those for the discontinuous gradients. Examples of separations performed on continuous gradients include the purification of Leydig cells (31), lactotrophs (19), bone marrow cells (52), intestinal epithelial cells (18), marine microalgae (28, 60) and chloroplasts (49, 58, 76, 88, 109). Preformed self-generated gradients Preforming a gradient by centrifugation can be a convenient alternative to using a gradient maker or pump. As described earlier, Percoll will sediment when subjected to significant g-forces (i.e. >10,000 x g). When preforming a gradient, SIP is diluted to a density that lies in the middle of the range in which maximum resolution is required. Two centrifuge tubes are filled with gradient material - one for the experiment and one containing Density Marker Beads. This second tube serves both as a counterbalance and as an external method for monitoring the gradient. The tubes are centrifuged in an angle-head rotor (e.g. 30,000 x g for 15 minutes), and the gradient forms isometrically around the starting density (Figure 4). The relatively "flat" region of the gradient should encompass the range required for maximum resolution of the target cells. This can be confirmed by observing the shape of the gradient in the tube containing the Density Marker Beads. The gradient becomes progressively steeper with time. It has been shown that the shape of the gradient is approximately linear related to the total g-force and time of the centrifugation (22). After forming the gradient, isopycnic banding of cells can be accomplished by low-speed centrifugation for 15-20 minutes at 400 x g. If an estimate of cell density is required, a volume equal to that of the cell suspension is layered on top of the tube containing the Density Marker Beads,. This serves as both a way to estimate cell density and as a counter-balance. Gradients formed in situ The sedimentation coefficients of subcellular particles and viruses are usually too low to allow banding on preformed gradients at low g-forces. Therefore, it is often convenient to mix the suspension of biological particles with Percoll and to band the particles on a gradient formed in situ. Gradients of Percoll formed by centrifugation are metastable - i.e. they will change continuously during high speed centrifugation. The rate of sedimentation of the colloid is slow enough to allow the banding of small viruses and cell organelles with "S" values >60S as the gradient is formed in situ. A common method for forming gradients in situ is to prepare a SIP, using 9 parts of Percoll to 1 part of 2.5 M sucrose. The SIP is then diluted to the desired density using 0.25 M sucrose. (Although sucrose is typically used to make in situ gradients, cell culture media can also be used). When mixing the sample directly with gradient material, the effect on the overall density of the Percoll solution can be calculated from the formula on page 13. Premixing of the sample with the gradient material is convenient when it is desirable to accurately measure the buoyant density of the particles. However, it may be better to layer the experimental sample on top of the gradient material, particularly in cases where it is desirable to separate subcellular particles from soluble proteins. The soluble proteins will remain in the buffer layer above the gradient and subcellular particles will separate in the Percoll gradient in situ. Centrifugation must be carried out in an angle-head rotor. A balance tube containing Density Marker Beads in place of experimental sample is used to monitor the gradient. An appropriate model experiment similar to the one described on page 22, should be carried out first to establish the gradient formation characteristics of the rotor to be used
Maximum sample loading There are no standard rules governing the maximum quantity of cells or subcellular material which can be separated on gradients of Percoll.For subcellular fractionation,successful purification can be achieved with a total loading of 1-5 mg of protein in a samlpe volume of 0.5 ml on 10 ml of gradient material(Pertoft,personal communication). A model experiment to standardize conditions angle the rotor use to enable you to establish a series of gradient curves for a particular rotor and tubes,and can be used as a reference for all future experiments. The example chosen is for 10 ml gradients,but this may be scaled up for larger tube sizes. 1.Mix 49.5 ml of Percoll with 5.5 ml of 1.5 M NaCl to make a SIP. 2.Mix SIP from step 1 with 0.15 M NaCl to make a series of 10 ml experime ta samples (total centrifuge tube size =13.5 ml)as shown in the following table: Tube No. 12345678910 Percoll (SIP)(ml) 10987654321 0.15 M NaCI (ml) 123456789 3.Add 10 pl of a suspens sion of each type of Density Marker Beads to each tube according to the instructions supplied in the pack 4.Balance and cap the tubes,and mix them by inverting several times. 5.Place the tubes in the angle-head rotor(if there are only 8 spaces,omit tubes 1 and 10). 6.Centrifuge at 30,000 x g for 15 minutes. 7.Carefully remove the tubes and using millimeter graph paper,measure to the nearest 0.5 mm the distance of each band from the bottom of the tube. 8.Plot the gradient shape for each tube by calibrating each band with the exact boyant density for each Marker Bead. 9.Re-mix the contents of each tube by inversion and repeat the centrifugation,this time using 60,000 x g for 15 minutes. 10.Measure the gradients and plot the results as before.calculate the exact density of the dilution using the formula (s 131i of curves enerated n M NC es 9 and 10 show typical examples of a series can be rep eated using Percoll in 0.25 M su 0.00for mimute followed By 100.0or miue in this case,running conditions 22
22 Maximum sample loading There are no standard rules governing the maximum quantity of cells or subcellular material which can be separated on gradients of Percoll. For subcellular fractionation, successful purification can be achieved with a total loading of 1-5 mg of protein in a samlpe volume of 0.5 ml on 10 ml of gradient material (Pertoft, personal communication). A model experiment to standardize conditions The exact shape and range of gradients formed during centrifugation is influenced by the model and angle of the rotor used, and by the size of the centrifuge tubes. The following experiment is designed to enable you to establish a series of gradient curves for a particular rotor and tubes, and can be used as a reference for all future experiments. The example chosen is for 10 ml gradients, but this may be scaled up for larger tube sizes. 1. Mix 49.5 ml of Percoll with 5.5 ml of 1.5 M NaCl to make a SIP. 2. Mix SIP from step 1 with 0.15 M NaCl to make a series of 10 ml experimental samples (total centrifuge tube size = 13.5 ml) as shown in the following table: 3. Add 10 µl of a suspension of each type of Density Marker Beads to each tube according to the instructions supplied in the pack. 4. Balance and cap the tubes, and mix them by inverting several times. 5. Place the tubes in the angle-head rotor (if there are only 8 spaces, omit tubes 1 and 10). 6. Centrifuge at 30,000 x g for 15 minutes. 7. Carefully remove the tubes and using millimeter graph paper, measure to the nearest 0.5 mm the distance of each band from the bottom of the tube. 8. Plot the gradient shape for each tube by calibrating each band with the exact boyant density for each Marker Bead. 9. Re-mix the contents of each tube by inversion and repeat the centrifugation, this time using 60,000 x g for 15 minutes. 10. Measure the gradients and plot the results as before. Calculate the exact density of the dilution using the formula (see page 13). Figures 9 and 10 show typical examples of a series of curves generated using Percoll in 0.15 M NaCl. The experiment can be repeated using Percoll in 0.25 M sucrose; in this case, running conditions should be 50,000 x g for 25 minutes followed by 100,000 x g for 25 minutes. Tube No. 1 2 3 4 5 6 7 8 9 10 Percoll (SIP) (ml) 10 9 8 7 6 5 4 3 2 1 0.15 M NaCl (ml) - 1 2 3 4 5 6 7 8 9