ANALYTICAL
BIOCHEMISTRY
87,
SHORT
243-248
(1978)
COMMUNICATIONS
An Alternate Method for the Preparative Velocity Sedimentation of Cells at Unit Gravity In this report we describe a technique which offers an alternate experimental approach among those already available (l-8) for the preparative velocity sedimentation of mammalian cells at lg. The method utilizes a commercially available (Buchler Poly-Prep 200) apparatus which has been used for cell electrophoresis experiments (9). The special features of this technique are (a) the apparatus used provides rapid thermal equilibration by means of both external and internal cooling of the separation chamber, (b) it requires the handling of small volumes of solutions (i.e., 100 to 150 ml) which shortens the time of collection, and (c) it allows the collection of fractions without mixing, by the continuous formation of a sharp interface between the chase solution and the density gradient. The latter is accomplished by pumping the chase solution in the column at a lower velocity than that of the gradient being pumped out (9). The Buchler Poly-Prep 200 (Buchler Instruments, Fort Lee, N. J.) used for the velocity sedimentation experiments is shown in Fig. 1. The apparatus has a glass column with an outer cooling jacket and an inner cooling glass piece which is inserted into the center of the column. An annulus (hollow cylinder) is thus produced having a cross-sectional area of 17.6 cm2. The gradient formation and subsequent cell fractionation takes place within this annulus. All glass surfaces in contact with the cells are washed thoroughly by immersion in PEX (Peck’s Products Co., St. Louis, MO.) and rinsed several times with hot tap water, followed by three distilled-water rinses and air drying. The annulus is subsequently siliconized with 1% solution of Siliclad (Clay Adams, Parsippany, N. Y.) in water. The column is filled with the siliconizing solution, drained slowly (20 min), rinsed extensively with distilled water, and air dried. The arrangement of the BSA/PBS solutions in the Buchler Poly-Prep 200 column is as follows. The bottom solution, 3% bovine serum albumin (BSA) in phosphate buffered saline (PBS), acts as a cushion for the density gradient, so that the lower end of the gradient is just at the orifice of the elution capillary. It also serves as a “chase” solution which is pumped from the reservoir into the bottom of the column during elution of the fractions. The BSA/PBS gradient (1 to 2% BSA) covers the density range 243
0003.2697/78/0871-0243$02.00/O Copyright 0 1978 by Academic Press. Inc. Ail rights of reproduction in any form reserved
244
SHORT COMMUNICATIONS ECUTION
OUT-
+-COOLANT COOLANT
IN
I 4-b
OUT+a
+-UPPER
UPPER
SOLUTION
P --E
OUT-
SOLUTION
LECTROOE
tCOOLANT
A’ SEPARATION
BOTTOM
UPPER
COLUMNCOOLANT
IN
SOLUTION
IN \
IN
OUT SOLUTION
SAMPLE
I.
‘k
OENSiYY
GRADIENT
BOTTOM
SOLUTION
OUS
GLASS
MEMBRANE
-0WER
L(MER
SOLUTION
LOWER
SOLUTION
IN
FIG. 1. Schematic diagram of the Buchler Poly-Prep 200 employed in velocity sedimentation work.
of 1.0099 to 1.0123 g/cm3 at 4°C. The density of the gradient is thus much lower than the density of mammalian cells which usually varies from 1.060 to 1.090 g/cm”. Pulse loading of the cells on top of this gradient allows them to sediment toward the bottom of the column. Details of the experimental procedure are as follows. The two parts of the lower buffer-electrode section are assembled and leveled. The column assembly is placed on top of the lower buffer section-being careful to align the red dots-and securely fastened with four sliding adjustable clamps. Subsequently, the internal glass cooling tube containing the central elution capillary is inserted into the column assembly and adjusted so that its lower surface is approximately 2.5 cm above the porous glass membrane. The coolant outport of the assembly and the coolant
SHORTCOMMUNICATIONS
245
inport of the internal glass cooling tube are connected. A cooler adjusted to 4°C is connected to the coolant inport of the column assembly and to the coolant outport of the internal glass cooling tube, and circulation is begun. Bottom solution (3% BSA, -80 ml) is introduced into the column assembly to the level of the lower surface of the internal glass cooling piece through the bottom-solution inport, filling the circular channel and entering the column assembly interior through the six ports of the lower buffer electrode section. A multiple-entry port is used to block the side tube of the column assembly. A tubing assembly consisting of 45 cm of l.O-mm (i.d.) Teflon tubing, lo-cm of 2-mm (i.d.) Tygon tubing, and 2 cm of 4-mm (i.d.) Tygon tubing is inserted through a small hole in this port so that only the Teflon tubing is located in the column interior. This tubing assembly is used to layer the density gradient (1 to 2% BSA), sample (0.5% BSA), and upper solution (PBS), in that order. The tubing assembly is filled with bottom solution, clamped, and attached to a gradient maker (LKB 8 121, LKB Produkter AB, Bromma, Sweden) containing 50 ml of the dense solution (2% BSA) and 50 ml of the light solution (1% BSA). The density gradient (100 ml) is formed in the column assembly at a rate of 1 ml/min. The tubing assembly is clamped just before it empties and is attached to a cooled (4°C) lo-ml syringe body in which is placed the sample solution (0.5% BSA, 10 ml), containing not more than 5 x lo6 cells/ml. The sample solution is carefully layered (1 ml/min) on top of the gradient. Just as the last of the sample solution leaves the cooled syringe, 10 ml of phosphate buffered saline is quickly placed in the syringe body and allowed to flow at the same rate on top of the sample solution. Time zero is designated as that time at which one-half ofthe sample solution has been layered on the gradient. In the layering of all solutions, the tubing assembly should always be clamped shut just as the last of the solution followed by an air pocket migrates about 1 cm into the tubing assembly. Failure to do this will cause the remaining contents (-2 ml) to flow at great speed into the column, thus mixing the solutions already in place. In addition, the tubing assembly should always contain fluid when introduction of the solutions into the column is begun-to permit smooth initiation of the flow. After the desired time of velocity sedimentation, fractions are collected in the following manner. The bottom solution is pumped at 1.0 ml/min (Technicon yellow/yellow tubing) into the bottom solution inport. Simuitaneously the gradient is pumped out through the elution capillary at a rate of 2.9 ml/min (Technicon purple/black tubing) and collected into - l.S-ml fractions for subsequent analysis. A typical fractionation of human blood cells by velocity sedimentation at lg is shown in Fig. 2. The open circles represent the distribution of the white cells [mononuclear (MN) plus polymorphonuclear (PMN) cells] and the solid circles the erythrocytes (RBC) and platelets (Pit). The curves were normalized by computer to the highest peak of each distribution.
246
SHORT COMMUNICATIONS
FRACTION
NUMBER
FIG. 2. Velocity sedimentation at lg of human blood ceils. Open circles represent the distribution of white blood cells (WBC) and solid circles represent the distribution of erythrocytes (RBC) and platelets (Pit). Each distribution has been normalized to the highest peak by computer. MN denotes mononuclear white cells and PMN polymorphonuclear cells. Differential counts were performed on Wright-stained slides.
The dotted lines indicate the fractions where highly enriched (90-100%) populations of a particular type of cell were found. Thus, the polymorphonuclear cells were distinctly separated from the mononuclear cells (lymphocytes and monocytes), erythrocytes, and platelets. However, separation of mononuclear cells from erythrocytes was not achieved. Highly enriched platelet fractions relatively free of other cells were obtained. The above results are in general agreement with the data presented by Brubaker and Evans (10) on the fractionation of human blood cells using the large cylindrical chamber. The electronic volume distribution of selected fractions recorded with a Coulter sizing apparatus is shown in Fig. 3. It may be seen that the large-volume cells (indicated by a higher channel number) are found primarily toward the lower part of the column (small fraction number). The small-volume cells are found in the opposite direction. Intermediatesize cells are present in the middle fractions. However, the transition from small- to intermediateto large-volume cells is gradual, which indicates overlapping of distributions in certain fractions. The peaks in Fig. 3 represent the following cell fractions: Channel 5, platelets; Channel 12, erythrocytes; Channel 30, mononuclear cells; Channel 60, polymorphonuclear cells. In conclusion, we have described a technique which offers an alternate experimental approach in carrying out preparative velocity-sedimentation fractionation of mammalian cells at lg. The method utilizes a commercially available and inexpensive apparatus which should facilitate work involving physical separation of cells. The same apparatus can be used for preparative density-gradient electrophoresis of cells in an isoosmolar medium (9).
SHORT
COMMUNICATIONS
247
248
SHORT COMMUNICATIONS
ACKNOWLEDGMENTS This work was supported by NC1 Contract No. NOl-CB-43928 and ONR Contract No. NOO014-70-C-0111. The skillful technical assistance of Carolyn Wish is acknowledged with thanks.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Mel, H. (1963) Nature (London) 200, 423-425. Peterson, E. A., and Evans, W. H. (1967) Nature (London) 214, 824-825. Miller, R. G., and Phillips, R. A. (1969) J. Cell Physiol. 73, 191-202. Tom, R., Hampe, A., and Sauerbrey, G. (1969) Z. Gesamfe Exp. Med. 151, 331-349. Nakeff, A., and Maat, B. (1974) Blood 43, 591-595. Tulp, A., and Bont, W. S. (1975) Analyt. Biochem. 67, 11-21. Bont, W. S., and Hilgers, J. H. M. (1977) Prep. Biochem. 7, 45-60. Zeiller, K., Hansen, E., Leihener, D., Pascher, G., and Wirth, H. (1976) HoppeSeyler’s Z. Physiol. Chem. 357, 1309-1319. 9. Griffith, A. L., Catsimpoolas, N., and Wortis, H. H. (1975) Life Sci. 16, 1693-1702. 10. Brubaker, L. H., and Evans, W. H. (1969) J. Lab. Clin. Med. 73, 1036-1041. N. CATSIMPOOLAS A. L. GRIFFITH E. M. SKRABUT Biophysics Department Cambridge,
Laboratory of Nutrition Mass. 02139
and Food Science,
MIT,
C. R. VALERI Naval Blood Research Boston, Mass 02118 Received November
Laboratory 24, 1976; accepted
January
27, 1978
BIOCHEMISTRY
87,
SHORT
243-248
(1978)
COMMUNICATIONS
An Alternate Method for the Preparative Velocity Sedimentation of Cells at Unit Gravity In this report we describe a technique which offers an alternate experimental approach among those already available (l-8) for the preparative velocity sedimentation of mammalian cells at lg. The method utilizes a commercially available (Buchler Poly-Prep 200) apparatus which has been used for cell electrophoresis experiments (9). The special features of this technique are (a) the apparatus used provides rapid thermal equilibration by means of both external and internal cooling of the separation chamber, (b) it requires the handling of small volumes of solutions (i.e., 100 to 150 ml) which shortens the time of collection, and (c) it allows the collection of fractions without mixing, by the continuous formation of a sharp interface between the chase solution and the density gradient. The latter is accomplished by pumping the chase solution in the column at a lower velocity than that of the gradient being pumped out (9). The Buchler Poly-Prep 200 (Buchler Instruments, Fort Lee, N. J.) used for the velocity sedimentation experiments is shown in Fig. 1. The apparatus has a glass column with an outer cooling jacket and an inner cooling glass piece which is inserted into the center of the column. An annulus (hollow cylinder) is thus produced having a cross-sectional area of 17.6 cm2. The gradient formation and subsequent cell fractionation takes place within this annulus. All glass surfaces in contact with the cells are washed thoroughly by immersion in PEX (Peck’s Products Co., St. Louis, MO.) and rinsed several times with hot tap water, followed by three distilled-water rinses and air drying. The annulus is subsequently siliconized with 1% solution of Siliclad (Clay Adams, Parsippany, N. Y.) in water. The column is filled with the siliconizing solution, drained slowly (20 min), rinsed extensively with distilled water, and air dried. The arrangement of the BSA/PBS solutions in the Buchler Poly-Prep 200 column is as follows. The bottom solution, 3% bovine serum albumin (BSA) in phosphate buffered saline (PBS), acts as a cushion for the density gradient, so that the lower end of the gradient is just at the orifice of the elution capillary. It also serves as a “chase” solution which is pumped from the reservoir into the bottom of the column during elution of the fractions. The BSA/PBS gradient (1 to 2% BSA) covers the density range 243
0003.2697/78/0871-0243$02.00/O Copyright 0 1978 by Academic Press. Inc. Ail rights of reproduction in any form reserved
244
SHORT COMMUNICATIONS ECUTION
OUT-
+-COOLANT COOLANT
IN
I 4-b
OUT+a
+-UPPER
UPPER
SOLUTION
P --E
OUT-
SOLUTION
LECTROOE
tCOOLANT
A’ SEPARATION
BOTTOM
UPPER
COLUMNCOOLANT
IN
SOLUTION
IN \
IN
OUT SOLUTION
SAMPLE
I.
‘k
OENSiYY
GRADIENT
BOTTOM
SOLUTION
OUS
GLASS
MEMBRANE
-0WER
L(MER
SOLUTION
LOWER
SOLUTION
IN
FIG. 1. Schematic diagram of the Buchler Poly-Prep 200 employed in velocity sedimentation work.
of 1.0099 to 1.0123 g/cm3 at 4°C. The density of the gradient is thus much lower than the density of mammalian cells which usually varies from 1.060 to 1.090 g/cm”. Pulse loading of the cells on top of this gradient allows them to sediment toward the bottom of the column. Details of the experimental procedure are as follows. The two parts of the lower buffer-electrode section are assembled and leveled. The column assembly is placed on top of the lower buffer section-being careful to align the red dots-and securely fastened with four sliding adjustable clamps. Subsequently, the internal glass cooling tube containing the central elution capillary is inserted into the column assembly and adjusted so that its lower surface is approximately 2.5 cm above the porous glass membrane. The coolant outport of the assembly and the coolant
SHORTCOMMUNICATIONS
245
inport of the internal glass cooling tube are connected. A cooler adjusted to 4°C is connected to the coolant inport of the column assembly and to the coolant outport of the internal glass cooling tube, and circulation is begun. Bottom solution (3% BSA, -80 ml) is introduced into the column assembly to the level of the lower surface of the internal glass cooling piece through the bottom-solution inport, filling the circular channel and entering the column assembly interior through the six ports of the lower buffer electrode section. A multiple-entry port is used to block the side tube of the column assembly. A tubing assembly consisting of 45 cm of l.O-mm (i.d.) Teflon tubing, lo-cm of 2-mm (i.d.) Tygon tubing, and 2 cm of 4-mm (i.d.) Tygon tubing is inserted through a small hole in this port so that only the Teflon tubing is located in the column interior. This tubing assembly is used to layer the density gradient (1 to 2% BSA), sample (0.5% BSA), and upper solution (PBS), in that order. The tubing assembly is filled with bottom solution, clamped, and attached to a gradient maker (LKB 8 121, LKB Produkter AB, Bromma, Sweden) containing 50 ml of the dense solution (2% BSA) and 50 ml of the light solution (1% BSA). The density gradient (100 ml) is formed in the column assembly at a rate of 1 ml/min. The tubing assembly is clamped just before it empties and is attached to a cooled (4°C) lo-ml syringe body in which is placed the sample solution (0.5% BSA, 10 ml), containing not more than 5 x lo6 cells/ml. The sample solution is carefully layered (1 ml/min) on top of the gradient. Just as the last of the sample solution leaves the cooled syringe, 10 ml of phosphate buffered saline is quickly placed in the syringe body and allowed to flow at the same rate on top of the sample solution. Time zero is designated as that time at which one-half ofthe sample solution has been layered on the gradient. In the layering of all solutions, the tubing assembly should always be clamped shut just as the last of the solution followed by an air pocket migrates about 1 cm into the tubing assembly. Failure to do this will cause the remaining contents (-2 ml) to flow at great speed into the column, thus mixing the solutions already in place. In addition, the tubing assembly should always contain fluid when introduction of the solutions into the column is begun-to permit smooth initiation of the flow. After the desired time of velocity sedimentation, fractions are collected in the following manner. The bottom solution is pumped at 1.0 ml/min (Technicon yellow/yellow tubing) into the bottom solution inport. Simuitaneously the gradient is pumped out through the elution capillary at a rate of 2.9 ml/min (Technicon purple/black tubing) and collected into - l.S-ml fractions for subsequent analysis. A typical fractionation of human blood cells by velocity sedimentation at lg is shown in Fig. 2. The open circles represent the distribution of the white cells [mononuclear (MN) plus polymorphonuclear (PMN) cells] and the solid circles the erythrocytes (RBC) and platelets (Pit). The curves were normalized by computer to the highest peak of each distribution.
246
SHORT COMMUNICATIONS
FRACTION
NUMBER
FIG. 2. Velocity sedimentation at lg of human blood ceils. Open circles represent the distribution of white blood cells (WBC) and solid circles represent the distribution of erythrocytes (RBC) and platelets (Pit). Each distribution has been normalized to the highest peak by computer. MN denotes mononuclear white cells and PMN polymorphonuclear cells. Differential counts were performed on Wright-stained slides.
The dotted lines indicate the fractions where highly enriched (90-100%) populations of a particular type of cell were found. Thus, the polymorphonuclear cells were distinctly separated from the mononuclear cells (lymphocytes and monocytes), erythrocytes, and platelets. However, separation of mononuclear cells from erythrocytes was not achieved. Highly enriched platelet fractions relatively free of other cells were obtained. The above results are in general agreement with the data presented by Brubaker and Evans (10) on the fractionation of human blood cells using the large cylindrical chamber. The electronic volume distribution of selected fractions recorded with a Coulter sizing apparatus is shown in Fig. 3. It may be seen that the large-volume cells (indicated by a higher channel number) are found primarily toward the lower part of the column (small fraction number). The small-volume cells are found in the opposite direction. Intermediatesize cells are present in the middle fractions. However, the transition from small- to intermediateto large-volume cells is gradual, which indicates overlapping of distributions in certain fractions. The peaks in Fig. 3 represent the following cell fractions: Channel 5, platelets; Channel 12, erythrocytes; Channel 30, mononuclear cells; Channel 60, polymorphonuclear cells. In conclusion, we have described a technique which offers an alternate experimental approach in carrying out preparative velocity-sedimentation fractionation of mammalian cells at lg. The method utilizes a commercially available and inexpensive apparatus which should facilitate work involving physical separation of cells. The same apparatus can be used for preparative density-gradient electrophoresis of cells in an isoosmolar medium (9).
SHORT
COMMUNICATIONS
247
248
SHORT COMMUNICATIONS
ACKNOWLEDGMENTS This work was supported by NC1 Contract No. NOl-CB-43928 and ONR Contract No. NOO014-70-C-0111. The skillful technical assistance of Carolyn Wish is acknowledged with thanks.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Mel, H. (1963) Nature (London) 200, 423-425. Peterson, E. A., and Evans, W. H. (1967) Nature (London) 214, 824-825. Miller, R. G., and Phillips, R. A. (1969) J. Cell Physiol. 73, 191-202. Tom, R., Hampe, A., and Sauerbrey, G. (1969) Z. Gesamfe Exp. Med. 151, 331-349. Nakeff, A., and Maat, B. (1974) Blood 43, 591-595. Tulp, A., and Bont, W. S. (1975) Analyt. Biochem. 67, 11-21. Bont, W. S., and Hilgers, J. H. M. (1977) Prep. Biochem. 7, 45-60. Zeiller, K., Hansen, E., Leihener, D., Pascher, G., and Wirth, H. (1976) HoppeSeyler’s Z. Physiol. Chem. 357, 1309-1319. 9. Griffith, A. L., Catsimpoolas, N., and Wortis, H. H. (1975) Life Sci. 16, 1693-1702. 10. Brubaker, L. H., and Evans, W. H. (1969) J. Lab. Clin. Med. 73, 1036-1041. N. CATSIMPOOLAS A. L. GRIFFITH E. M. SKRABUT Biophysics Department Cambridge,
Laboratory of Nutrition Mass. 02139
and Food Science,
MIT,
C. R. VALERI Naval Blood Research Boston, Mass 02118 Received November
Laboratory 24, 1976; accepted
January
27, 1978
