(p-p) Vy=V; (p-p) where v P y of di d density of 0.25 M sucrose is -1.032 g/ml) density of diluted solution produced in g/ml Example: To dilute 55 ml of SIP to a final density of 1.07 g/ml determine the amount of 0.15 M NaCl required. 1.123-1.07 Volume of 0.15 M NaCl required =55 x 1.07-1.0046 =44.6ml desired. ever, slight variat ons in volumes and densi density.For determining actual dens ies,we recommend measuring the final density of Percoll solutions using a densitometer or refractometer (see page 25). Note:The graph shown in Figure 5 can also be used as an pirical guide to the dens itu of solutions on of SIp This graph refers to th dilut vhe 1.12 90 /V)L coll osmotic saline or sucrose oid con on,it is t山 110 preferable to refer to the actual density of the working solution (or to state SIP)rather than 1.08. 25 to refer to the solution as a percentage of Percoll in iso-osmotic saline or sucrose.This is particularly 1.06 important when using the one-step dilution procedure described below,where a working solution of known density is obtained by diluting Percoll (undiluted)plus ntrated salts o 1.02 sucrose to a final volume vith distilled wate i0200050607000160 Percoll (SIP)with is
13 % of Stock Isotonic Percoll (SIP) in final solution Dilution with 0.25 M Sucrose Dilution with 0.15 M NaCl 10 20 30 40 50 60 70 80 90 100 1.02 1.04 1.06 1.08 1.10 ro =1.135 ro =1.130 ro =1.125 ro =1.135 ro =1.130 ro =1.125 1.12 1.14 Density g/ml where Vy = volume of diluting medium in ml Vi = volume of SIP in ml ri = density of SIP in g/ml ry = density of diluting medium in g/ml (density of 0.15 M NaCl is ~1.0046 g/ml) * (density of 0.25 M sucrose is ~1.032 g/ml)* r = density of diluted solution produced in g/ml Example: To dilute 55 ml of SIP to a final density of 1.07 g/ml, determine the amount of 0.15 M NaCl required. 1.123 - 1.07 Volume of 0.15 M NaCl required = 55 x 1.07 - 1.0046 = 44.6 ml The above formula is useful for achieving densities that will be very close to the actual density desired. However, slight variations in volumes and densities of diluting media will affect final density. For determining actual densities, we recommend measuring the final density of Percoll solutions using a densitometer or refractometer (see page 25). * from CRC Handbook of Chemistry and Physics, 67th edition (1986-1987), CRC Press, D253 and D262. Figure 5. Dilution of Stock Isotonic Percoll (SIP) with iso-osmotic saline or sucrose solution. Po is the density of the Percoll (undiluted). SIP is prepared as described on page 12. The calibration lines shown are for guidance only. For accurate density measurements, refer to the formula given in the text. (Work from Amersham Biosciences, Uppsala, Sweden.) Note: The graph shown in Figure 5 can also be used as an empirical guide to the density of solutions produced by diluting SIP with 0.15 M saline or 0.25 M sucrose. This graph refers to the dilution of SIP where SIP is 90% (v/v) undiluted Percoll osmotically adjusted by addition of 10% (v/v) saline or sucrose. To avoid confusion, it is therefore preferable to refer to the actual density of the working solution (or to state % SIP) rather than to refer to the solution as a percentage of Percoll in iso-osmotic saline or sucrose. This is particularly important when using the one-step dilution procedure described below, where a working solution of known density is obtained by diluting Percoll (undiluted) plus concentrated salts or sucrose to a final volume with distilled water. (ri -r) Vy = Vi (r-ry)
The one-step procedure for diluting Percoll Percoll (undiluted)may de diluted directly to make a final working solution of known density by the following procedure.In a measuring cylinder,add 1.5 M NaCl or 2.5 M sucrose to 1/10 of the final desired volume (e.g.10 ml for 100 ml of working solution).To this,add the required volume of Percoll(undiluted),calculated using the formula shown below.Make up to the final volume with distilled water. V。=VP-0.1po-0.9 P。-1 where V volume of Percoll(undiluted)(ml) (g/ml) Po ndil ted)(g/ml) see Certifi 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 additions) Example: To prepare 100 ml of working solution of Percoll of density 1.07 g/ml in 0.15 M NaCl.To 10 ml of 1.5 M NaCl,add 1.07-0.1058-0.9 Volume of Percoll required =100x 0.13 .ml(if Percoll density is 1.10 The above formula is useful for achieving densities that will be very close to the actual density desired.However,slight variations in volumes and densities of diluting media will affect final density.For determining highly accurate densities,we recommend measuring the final density of Percoll solutions using a densitometer or refractometer (see page 25). Graphs similar to the one shown in Figure 5 can be drawn to relate the volume of Percoll(undiluted) to the final density. 14
14 where Vo = volume of Percoll (undiluted) (ml) V = volume of the final working solution (ml) r = desired density of the final solution (g/ml) ro = density of Percoll (undiluted) (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 additions) The one-step procedure for diluting Percoll Percoll (undiluted) may de diluted directly to make a final working solution of known density by the following procedure. In a measuring cylinder, add 1.5 M NaCl or 2.5 M sucrose to 1/10 of the final desired volume (e.g. 10 ml for 100 ml of working solution). To this, add the required volume of Percoll (undiluted), calculated using the formula shown below. Make up to the final volume with distilled water. The above formula is useful for achieving densities that will be very close to the actual density desired. However, slight variations in volumes and densities of diluting media will affect final density. For determining highly accurate densities, we recommend measuring the final density of Percoll solutions using a densitometer or refractometer (see page 25). Graphs similar to the one shown in Figure 5 can be drawn to relate the volume of Percoll (undiluted) to the final density. Example: To prepare 100 ml of working solution of Percoll of density 1.07 g/ml in 0.15 M NaCl. To 10 ml of 1.5 M NaCl, add 1.07 - 0.1058 - 0.9 Volume of Percoll required = 100 x 0.13 = 49.4 ml (if Percoll density is 1.130 g/ml) and make up to 100 ml with distilled water. r - 0.1r10 - 0.9 Vo = V ro - 1
Diluting Percoll to a desired osmolality To make isotonic Percoll for most mammalian cells,it is common to dilute parts of Percoll (undiluted) with 1 part of 1.M NaClor ion.This Stock Isotonic Percoll (SIP)is ther 6 eds.H weve while this edure has proved athe and does ot take into accou t th t of olid silica par present (i.e.that 1 stock conta ns a certain volum of solid sili making the tota aqueous volume less than 100 ml).Due to the volume occupied by silica,the electrolytes in the stock solution have a higher effective concentration than in physiological salt solution,and SIP made in this way will be hyperosmolal.Thus,determining the actual osmolality of the SIP has always been recommended. Vincent and Nadeau(555)discuss the problem elegantly and described an equation which can be used to calculate the number of parts of Percoll which should be added to one part of 10x concentrated physiological salt buffer to obtain a SIP of any desired osmolality.The authors determined the fraction of the total volume of a Percoll stock solution which is occupied by silica and thus deter- mined the ratio of volume of aqueous solution to that of total Percoll stock solution. 0e-0 Vp=V. (O;-Op) where Vp number of parts of Percoll to be added number of parts of solute concentrate (e.g.1.5 M NaCl)to be added osmolality of solute concentrate (e.g.1.5 M NaCl 2880 mOsm) desired osmolality R ratio of aqueous volume to total volume of Percoll(typically =0.85 for NaCl and 0.80 for sucrose) Op osmolality of Percoll undiluted (see Certificate of Analysis) The key variable in this equation is R,which is a measure of the real aqueous volume of a Percoll solution.The value of R is a function of the hydrodynamic volume occupied by the Percoll particles. This,in turn is a function of the ionic strength of the medium-that is,as ionic strength increases, hydrodynamic volume decreases.Thus,there is a difference in the R value of 1.5 M NaCl and 2.5 M sucrose. To obtain a SIP of osmolality 320 mOs/kg H2O adiusted with 1.5 M NaCl (i.e.10x concentrated physiological saline): 2880-320 V。=1 =10.04 .85(320-20) assuming: 2880= olality of 1.5 M NaCl (10 ated physi ogical saline) 20 osmolality of Percoll undiluted Therefore to obtain a SIP of 320 mOs/kg H2O,one would add 10 parts Percoll to 1 part 1.5 M NaCl
15 Diluting Percoll to a desired osmolality To make isotonic Percoll for most mammalian cells, it is common to dilute 9 parts of Percoll (undiluted) with 1 part of 1.5 M NaCl or 2.5 M sucrose solution. This Stock Isotonic Percoll (SIP) is then further diluted with physiological buffers according to needs. However, while this procedure has proved successful, it is rather simplistic and does not take into account the effect of having solid silica particles present (i.e. that 100 ml of Percoll stock contains a certain volume of solid silica, making the total aqueous volume less than 100 ml). Due to the volume occupied by silica, the electrolytes in the stock solution have a higher effective concentration than in physiological salt solution, and SIP made in this way will be hyperosmolal. Thus, determining the actual osmolality of the SIP has always been recommended. Vincent and Nadeau (555) discuss the problem elegantly and described an equation which can be used to calculate the number of parts of Percoll which should be added to one part of 10x concentrated physiological salt buffer to obtain a SIP of any desired osmolality. The authors determined the fraction of the total volume of a Percoll stock solution which is occupied by silica and thus determined the ratio of volume of aqueous solution to that of total Percoll stock solution. The key variable in this equation is R, which is a measure of the real aqueous volume of a Percoll solution. The value of R is a function of the hydrodynamic volume occupied by the Percoll particles. This, in turn is a function of the ionic strength of the medium - that is, as ionic strength increases, hydrodynamic volume decreases. Thus, there is a difference in the R value of 1.5 M NaCl and 2.5 M sucrose. To obtain a SIP of osmolality = 320 mOs/kg H2O adjusted with 1.5 M NaCl (i.e. 10x concentrated physiological saline): where Vp = number of parts of Percoll to be added Vc = number of parts of solute concentrate (e.g. 1.5 M NaCl) to be added Oc = osmolality of solute concentrate (e.g. 1.5 M NaCl = 2880 mOsm) Of = desired osmolality R = ratio of aqueous volume to total volume of Percoll (typically = 0.85 for NaCl and 0.80 for sucrose) Op = osmolality of Percoll undiluted (see Certificate of Analysis) Therefore to obtain a SIP of 320 mOs/kg H2O, one would add 10 parts Percoll to 1 part 1.5 M NaCl. 2880 - 320 Vp = 1 = 10.04 0.85 (320 - 20) assuming: 2880 = osmolality of 1.5 M NaCl (10x concentrated physiological saline) 20 = osmolality of Percoll undiluted Oc - Of Vp = Vc R(Of - Op)
The ratio of concentrated solute solution (i.e.1.5 M NaCl,etc.)to SIP is called R wheres R,= V。+V Using this formula Working wn density and osmolalitrc (undiluted)required to make a final V。=V P-RxP1o-(1-Rx) Po-1 where Voume of ero() fraction of total volume which is solute concentrate (i.e.1.5 M NaCl,etc.) density of Percoll undiluted(g/ml) (see Certificate of Analysis) 。 density of 1.5 M NaCl(1.058 g/ml),2.5 M sucrose (1.316g/ml,ctc. Thus,for 100 ml of SIP of osmolality =320 mOs/kg H2O adjusted with NaCl and density =1.07 g/ml 1.07-1/11x1.058-(1-1/11) Vo=100 =49.8ml 1.13-1 The final solution 16
16 The ratio of concentrated solute solution (i.e. 1.5 M NaCl, etc.) to SIP is called Rx where: The final solution contains 9.1 ml of 1.5 M NaCl (1/11 x 100 = 9.1), 49.8 ml Percoll undiluted and 34 ml (i.e. 100 - 58.9 = 41.1) of distilled water. Thus, for 100 ml of SIP of osmolality = 320 mOs/kg H2O adjusted with NaCl and density = 1.07 g/ml: where Vo = Volume of Percoll undiluted (ml) V = Volume of final working solution (ml) r = desired density of final working solution (g/ml) Rx = fraction of total volume which is solute concentrate (i.e. 1.5 M NaCl, etc.) ro = density of Percoll undiluted (g/ml) (see Certificate of Analysis) r10 = density of 1.5 M NaCl (1.058 g/ml), 2.5 M sucrose (1.316 g/ml), etc. Using this formula, one can calculate the amount of Percoll (undiluted) required to make a final working solution of known density and osmolality. r-Rxr10-(1-Rx) Vo = V ro -1 Vc Rx = Vp + Vc 1.07 - 1/11 x 1.058 - (1-1/11) Vo = 100 = 49.8 ml 1.13 - 1
Effects of osmolality on apparent buoyant density of cells and subcellular particles The very low osmolality of Percoll has facilitated the study of the interrelation of the 5115 30日 separation medium osmolality with the ticles 6 sho 1.10 20 m.The ppr nol/kg of the cells increases with increasing osmolal 1.05 ity,due to removal of water from the cells. The same effect has been observed with mitochondria(Figure 7)and with lysosomes (Table 1).Even small changes in osmolality cause a large change in the apparent buoyant 6. n of rat liver hepe s cells (35 x 10 cells densities of these organelles.The actual ith a der 065 alit recorded buoyant densities of particles banded of the in percoll gradients at physiological osmolality are therefore much more likely to cor espond ep.23 se exist nd publisher Particles are banded in sucrosorother f50 00 Fraction numbe g ne at4000
17 Effects of osmolality on apparent buoyant density of cells and subcellular particles Figure 6. Fractionation of rat liver hepatocytes cells (35 x 106 cells in a volume of 2 ml) on a self-generated Percoll gradient (8 ml solution with a density of 1.065 g/ml). The osmolality of the Percoll solution was varied by adding NaCl to 200 mOsm, 300 mOsm and 400 mOsm. Centrifugation was performed in a Beckman rotor 30.2 for 15 min at 35,000 x g at a temperature of 4 ºC. Density gradient determined using DMB (see p. 23). (27, reproduced by kind permission of the authors and publisher). Figure 7. The density distribution of mitochondria from rat liver cells after incubation in iso-osmotic buffer (red) and buffer containing 17.5% albumin (green). Centrifugations were performed in a Beckman 65 rotor (23º angle) for 30 minutes at 40,000 x g. (59, reproduced by kind permission of the authors and publisher). The very low osmolality of Percoll has facilitated the study of the interrelation of the separation medium osmolality with the apparent buoyant density of particles. Figure 6 shows the effects of banding rat liver hepatocytes in Percoll gradients having osmolalities of 200, 300 and 400 mOsmol/kg H2O (mOsm). The apparent buoyant density of the cells increases with increasing osmolality, due to removal of water from the cells. The same effect has been observed with mitochondria (Figure 7) and with lysosomes (Table 1). Even small changes in osmolality cause a large change in the apparent buoyant densities of these organelles. The actual recorded buoyant densities of particles banded in Percoll gradients at physiological osmolality are therefore much more likely to correspond to those existing in vivo, than when the particles are banded in sucrose or other centrifugation media. Distance from meniscus cm 1234 5 6 7 8 200 300 400 Hepatocytes (viable) Non-viable cells mOsm 10 20 30 1.10 1.05 1.15 Density gradient Density g/ml Number of cells x 106 Fraction number 0 0 5 10 15 20 10 20 30 40 50 70 60 1.10 1.05 1.15 Density g/ml Enzyme activity % of total Density gradient