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Separation of murine peritoneal macrophages using percoll density gradients
ELSEVIER
JOURNALOF IMMUNOLOGICAL METHODS Journal of ImmunologicalMethods 174 (1994) 53-59
Separation of murine peritoneal macrophages using percoll density gradients B e r n a r d V r a y *, N a t h a l i e P l a s m a n Laboratoire d'Imrnunologie (CP 615), Facult~ de M~decine, Universit~ Libre de Bruxelles, 808 route de Lennik, B-1070 Brussels, Belgium
Abstract
Macrophages harvested from the murine peritoneal cavity are functionally and morphologically heterogeneous. Here, we describe a procedure which permits the determination of specific cell densities using a continuous density gradient of Percoll (analytical step). Subsequently, discontinuous density gradients are used in routine (preparative step) to isolate all the cell subpopulations according to their actual specific density. This procedure has been successfully used for both mouse and rat peritoneal macrophages. Keywords: Murine peritoneal macrophage; Cell density; Cell separation; Continuous density Percoll gradient;
Discontinuous density Percoll gradient
1. Introduction
Due to their central role in the immune system, macrophages are widely used in experimental work. To study the various functions of these cells as well as to analyze their interactions with microorganisms, murine peritoneal macrophages (MPM) are frequently used since they are easy to harvest. However, their high functional and morphological heterogeneity (Bursuker and Goldman, 1983; De Bakker et al., 1985; Van Furth,
Abbreviations: MPM, mouse peritoneal macrophage; HBSS, Hanks' balanced salt solution. * Corresponding author. Tel.: 3225556260; Fax: 322555 6128.
1989; Leenen et al., 1990) and the presence of non-macrophagic cells (Meltzer, 1981; Kipps, 1989) can present problems. Consequently, the fractionation of peritoneal cells into functionally macrophage enriched subpopulations has triggered the development of various procedures based on Stokes' law: in a density gradient, the rate of cell sedimentation in a centrifugal field is zero when the cells encounter a medium of identical density (Pertoff, 1979). Such gradients are prepared either with Percoll (see below) or with Ficoll (Campbell et al., 1980; H o p p e r and Geczy, 1980; Miller and Morahan, 1982) or with bovine serum albumin (Fishman and Weinberg, 1979; Chapes and Tompkins, 1981; Tzehoval et al., 1981). The centrifugation step can also be omitted and cells are separated by velocity sedimenta-
0022-1759/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0022-1759(94)00155-P
54
B. Vray, N. Plasman /Journal of Immunological Methods 174 (1994) 53-59
tion (Reppun et al., 1979; Pelus et al., 1981; Wang et al., 1992). The Percoll density gradient technique has been widely adopted due to its ease of use. In this case, cell separations are based partly on Stokes' law and partly on the cell diameter (Lee, 1980; Coquette, 1989) or the cellular volume (Chandler et al., 1986a). In many studies, macrophages are separated on discontinuous (pre-formed) Percoll gradients. In this case, a relatively large number of density layers is arbitrarily fixed. Consequently, the number of cell subpopulations harvested depends on the number of the Percoll solutions which are overlayered in the tubes (Serio et al., 1979; Hester and Walker, 1981; Rasmussen et al., 1983; Murphy and Herscowitz, 1984; O'Neill et al., 1984; Thompson and Myrvik, 1985; BielefeldtOhmann and Babiuk, 1986; Oghiso, 1987; Ferro et al., 1987; Elias et al., 1987; Sutton et al., 1989; Sakai et al, 1991). On the whole, all these studies show a clear cut difference between cell subpopulations of low and high densities with respect to various functions. An improved cell separation can be obtained using continuous (or self-generated) density gradients of Percoll. A Percoll solution of a given density is centrifuged and high and low densities are distributed around this given density. An optimal cell separation is obtained when all the densities lie along a sigmoidal curve. Consequently, when a heterogeneous cell population is layered on a continuous gradient and centrifuged, numerous cell subpopulations are separated according to their actual specific density. Results obtained with this procedure underline the relationship between cell density and various functions which depend on the maturation stage of the cell (Gmelig-Meyling and Waldmann, 1980; Dauber et al., 1983; Shellito and Kaltreider, 1984). Linear continuous density gradients have also been used for the fractionation of rat alveolar macrophages (Chandler et al., 1986 a and b). Finally, a combination of self-generated and pre-formed gradients readily permits the harvesting of cell subpopulations enriched in macrophages (Coquette et al., 1988; Plasman and Vray, 1993).
The specific cell density of macrophages depends on the strain, the age and the sex of the mice or rats used. For this reason, it is recommended to first determine this specific cell density in an analytical step. This can be done by centrifuging simultaneously a continuous gradient containing coloured beads of well known density together with another gradient of the peritoneal cells to be studied. A direct comparison of the two tubes indicates the specific density of each cell subpopulation. Subsequently, a preparative step using a discontinuous density gradient permits all the cell subpopulations to be separated according to their actual specific density. The procedure that we describe here has been successfully used for both mouse and rat peritoneal macrophages. It could be readily adapted to other types of phagocyte.
2. Materials
Percoll (Pharmacia Fine Chemicals, Sweden) is a polyvinyl-pyrrolidone-coated silica which is supplied as a sterile colloidal suspension with characteristics (density: 1.130 + 0.005 g/ml; pH: 8.9 + 0.3 at 20°C; refraction index: 1.3540 + 0.005 at 20°C) which vary slightly from batch to batch. Other technical data can be found in the handbook Percoll, Methodology and Applications, which is available on request from the manufacturer. Hanks' balanced salt solution without Ca z+ and without Mg 2÷ (pH 7.4, HBSS, Gibco, Grand Island, New York). RPMI 1640 medium containing 10% of fetal calf serum, 100 U / m l penicillin and 100 /~g/ml streptomycin (Gibco). Mice: one mouse yields 2-3 × 106 resident peritoneal cells. 15 male or female mice weighing 25-30 g are required to harvest enough peritoneal cells (30 x 10 6) for one gradient. Cells are pooled and washed in a polypropylene (50 ml volume) conical tube (Becton Dickinson Labware, Falcon no. 2070). Continuous gradient: polystyrene, siliconized tubes forhigh speed centrifugation and adapted to a JA-30 rotor (centrifuge L2 Beckman Instrument Spinco, California, USA).
B. Vray, N. Plasman /Journal of Immunological Methods 174 (1994) 53-59
Discontinuous gradient: polystyrene, siliconized conical tubes (Becton Dickinson Labware, Falcon no. 2095).
3. Methods
55
3.2. Percoll solutions An iso-osmotic solution of Percoll of a given density (1/, working solution) is prepared by mixing sterile sodium chloride solution (1.5 M) and a stock solution of Percoll (1/o) according to the following formula:
3.1. Harvesting of peritoneal cells d - (0.1 × dl0 ) - 0.9 A mouse is killed with ether and pinned, ventral side up, on to a dissecting board under a sterile laminar flow. The abdomen skin is sterilized with ethanol and peeled back. Using a disposable syringe (5 ml) with an 20 gauge needle, peritoneal cells are harvested by two successive washings of the peritoneal cavity with pre-chilled HBSS. The peritoneal cells are gently introduced into a chilled polypropylene 50 ml conical tube (Falcon no. 2070). Then, all the peritoneal cells harvested from the 15 mice are pooled and washed twice in HBSS by centrifugation (300 x g for 10 min at 4°C). Cell pellets are resuspended in RPMI 1640 medium. After counting in a Thoma chamber, cells are adjusted to 1 × 106/ml.
A
1/o = V"
do- 1
where 1/o = volume (ml) of Percoll (stock solution); V = final volume (ml) of the working solution of Percoll; d = final density of the iso-osmotic working solution (g/ml); d o = density of the stock solution of Percoll (g/ml); dl0 = density of a 1.5 M NaCI solution (1.058 g/ml). Example. The density of the stock solution is d o = 1.130 g / m l (indicated on the bottle). To prepare 100 ml (1/) of an iso-osmotic solution of 1.070 g / m l (d), add 49.4 ml of Percoll (stock solution) to 10 ml of NaCI (1.5 M) in a sterile glass bottle and make up to 100 ml with sterile distilled water. Kept sterile, this stock solution
DENSITY (g/ml) 1.140-
I L____~
..- e ~
1.120 ,,./
/"
/ 1.100
./
Or"
O/ /
1.080
%../ II
/0
/
M
61
G;
R1
_.s."
,,,... ! f . , ' ~o
1.060
B1 01
B1
B; B;
1.040
.IO;
01
R;
1.020
V
R1
vR:
1.000 0
51
G; B;
v
3.5
DISTANCE IN CM'S FROM MENISCUS OF GRADIENT
I
II
III
Fig. 1. Density and cell distribution using a continuous Percoll gradient. A: calibration tubes were prepared with Percoll solutions (I: 1.114 g/ml; II: 1.090 g/ml; III: 1.036 g/ml) and mixed with coioured beads of known density. After centrifugation (40000 x g, 30 min, 4°C) in a JA-30 rotor (centrifuge L2 Beckman Instrument Spinco, California, USA), the distances between the meniscus and the coloured bands were plotted. B: distribution of coloured beads in the tubes after centrifugation. From the top to the bottom BI: blue, 1.016 g/ml; O1: orange, 1.033 g/ml; GI: green, 1.048 g/ml; RI: red, 1.062 g/ml; B2: blue, 1.076 g/ml; 02: orange, 1.088 g/ml; G2: green, 1.100 g/ml; R2: red, 1.118 g / m l and V: violet, 1.139 g/ml).
56 can be stored for the dark. The density of by measuring the tometer (Galileo, ture.
B. Vray, N. Plasman/ Journal of lmmunological Methods 174 (1994) 53-59 several days in the cold and in
CELL NUMBER / ml x 106
the Percoll solution is obtained refractive index with a refracMilan, Italy) at room tempera-
5-
lo
4-
3-
3.3. Preparation of an optimal continuous gradient profile
!
DENSITY ( g / m l )
1,139
1.118 1,100 1.088 1.076
2 1.062
The generation of a continuous gradient profile depends on various physical parameters. Before using the peritoneal cells, it is recommended to first test that the density gradient exhibits a sigmoidal profile. For this, a Percoll solution (1.090 g / m l , 40 ml) is prepared and chilled. To measure the distance between the meniscus and various coloured bands, an adhesive millimeter scale is attached to a calibration tube and 10/xl aliquots of the different coloured beads are added to 20 ml of the Percoll solution. The tube is then centrifuged (40000 x g, 30 min, 4°C) in a JA-30 rotor (centrifuge L2 Beckman Instrument Spinco, California, USA) and the density profile of the gradient is determined by plotting the distance between the meniscus and the different layers of coloured beads. As shown in Fig. 1 a, a sigmoidal profile is obtained with a density of 1.090 g / m l , ensuring an optimal cell separation: all the densities are distributed all along the top and the bottom of the tube (Fig. lb). By contrast, two other Percoll densities (1.036 and 1.114 g / m l ) give completely different profiles. 3.4. Determination o f specific cell densities with a continuous density gradient A pellet of peritoneal cells (30 x 106/ml) is mixed with 20 ml of Percoll solution (1.090 g / m l ) in a siliconized sterile polystyrene tube (experimental tube). A calibration tube prepared as described above is prepared and the two tubes are centrifuged together (40000 x g, 30 min, 4°C). Then, fractions of 250/zl are collected from the experimental tube, using an 18-gauge spinal needle connected to a peristaltic pump (model 2120 - LKB Instruments, Bromma, Sweden). Cells are washed twice in cold HBSS, resuspended in 250
1.048
1 234 0
•
1.033
7 i
I
1.016
I
2o
10
VOLUME ( ml )
I
0
315 DISTANCE IN CM'S FROM
MENISCUS OF GRADIENT
Fig. 2. Mouse peritoneal cells were harvested from male BALB/c mice. They were layered on the top of a continuous density gradient of Percoll (density 1.090 g/ml) and centrifuged. The figure illustrates the sigmoidal profile of the densities associated with the 12 subpopulations obtained after centrifugation.
/xl HBSS and counted. In our experimental model, 12 cell subsets are identified with male B A L B / c mice (Fig. 2) and 14 subsets with female mice. 3.5. Discontinuous density gradient To harvest large amounts of fractionated peritoneal cells, discontinuous gradients are prepared as above. 1 ml of a Percoll solution (1.117 g / m l ) is first poured into siliconized polystyrene tubes. Then, 1 ml of each specific cell density Percoll solution is layered from the heaviest at the bottom to the lighest at the top. Finally, 1 ml of Percoll (1.010 g / m l ) containing 3 × 107 cells/ml is layered on to the top of the gradient. The discontinuous gradient is centrifuged (400 X g, 4°C, 30 min) in a swinging bucket rotor (centrifuge J O U A N CR411, Saint Nazaire, France). Then, the various cell subsets are collected by suction with a siliconized sterile Pasteur pipette, washed twice in cold HBSS and resuspended to the appropriate cell concentration and can be used for microscopic observations, enzymatic, flow-cytometric and phagocytic
B. Vray, N. Plasman/Journal of Immunological Methods 174 (1994) 53-59
assays. The viability of cells is estimated by the trypan blue dye exclusion test.
4. Critical comments
Percoll appears to be the medium of choice for cell separation. Indeed, our results indicate a high percentage of cell viability together with the preservation of critical functions such as bacterial phagocytosis or eicosanoid synthesis (Coquette et al., 1988; Plasman and Vray, 1993). They also confirm previous work indicating that other cell functions are not affected (Serio et al., 1979; Gmelig-Meyleng and Waldmann, 1980; Pertoff et al., 1980). To inhibit spontaneous cell agregation, separations have to be performed with chilled materials and medium lacking Ca a+ and Mg 2+. Cell concentration is also critical and depends on the surface at the interface between Percoll layers. Thus, the choice of the tubes and their internal diameter is limiting for the cell suspension layered on the top of the gradient. Murine peritoneal cells include mainly macrophages (60-70%) with many lymphocytes (3040%) recovered in low density fractions. However, as a consequence of their large spectrum of densities, some lymphocytes are also present in macrophage enriched fractions. Most of these "contaminating" lymphocytes can be discarded by plating subsets for 2 h in culture dishes and allowing the macrophages to adhere. Subsequent washing with RPMI 1640 will remove most of the lymphocytes. Other non-macrophagic cells (neutrophils, erythrocytes and mast cells) are also encountered but at a very low percentage of the total population. They are found in high density fractions and at the bottom of the tube (Plasman and Vray, 1993). Fractionation of MPM into subpopulations depends on their specific cell density and cell diameter. These two parameters seem to be related to the maturation of the cell which does not necessarily correlate with physiological function. However, we have observed that the rate of bacterial phagocytosis, both antibody and complement mediated, and eicosanoid synthesis are related to macrophage density (Coquette et al., 1988). This
57
is also the case for rat alveolar macrophages (Chandler et al., 1986 a and b). Cell specific density depends on various parameters (e.g. the species, strain, age and sex of the animals used as well as the state of husbandry). Others parameters (choice of tubes, rotors and centrifuges) also influence the acccuracy of cell separations. Thus, it must be stressed that in order to obtain the continuous density gradient of Percoll permitting the best cell separations, it is necessary to test different densities together with centrifugation procedures of differing duration and speed.
5. Conclusion
Macrophages harvested from different anatomical sites are heterogeneous and this has hampered the analysis of many of their basic functions. Percoll density gradients offer the opportunity to eliminate most of the non-macrophagic cells and distribute macrophages of differing specific density according to their maturation stage and cell size.
Acknowledgments
The authors thank Mr. V. Vercruysse for excellent technical assistance, Mr. I. Mazza and Mr. N. Cardon for help in preparing the manuscript. This work was supported by grant "Action de Recherche Concert6e", U.L.B., 1991 and the Foundation M. M6rieux.
References
Bielefeldt-Ohmann, H. and Babiuk, L.A. (1986) Bovine alveolar macrophages: phenotypic and functional properties of subpopulations obtained by Percoll density gradient centrifugation. J. Leukocyte Biol. 39, 167. Bursuker, I. and Goldman, R. (1983) On the origin of macrophage heterogeneity. A hypothesis. J. Reticu|oendothel. Soc. 33, 207. Campbell, M.W., Sholley, M.M. and Miller, G.A. (1980) Macrophage heterogeneity in tumor resistance: cytostatic and cytotoxicactivityof Corynebacteriurn parvurn-activited
58
B. I/ray, N. Plasman /Journal of Immunological Methods 174 (1994) 53-59
and proteose peptone-elicited rat macrophages against Moloney sarcoma tumor cells. Cell. Immunol. 50, 153. Chandler, D.B., Fuller, W.C., Jackson, R.M. and Fulmer, J.D. (1986a) Studies of membrane receptors and phagocytosis in subpopulations of rat alveolar macrophages. Am. Rev. Respir. Dis. 133, 461. Chandler, D.B., Fuller, W.C., Jackson, R.M. and Fulmer, J.D. (1986b). Fractionation of rat alveolar macrophages by isopycnic centrifugation: morphological, cytocbemical, biochemical and functional properties. J. Leukocyte Biol. 39, 371. Chapes, S.K. and Tompkins, W.A.F. (1981) Distribution of macrophage cytotoxic and macrophage helper functions on BSA discontinuous gradients. J. Reticuloendothel. Soc. 30, 517. Coquette, A. (1989) Caractrrisation morphologique et fonctionnelle des sous-populations de macrophages. Th~se pr6sent6e pour l'obtention du grade de docteur en biologie m6dicale appliqu6e. Universit6 Libre de Bruxelles. Coquette, A., Boeynaems, J.M., Saint-Guillain, M. and Vray, B. (1988) Macrophage heterogeneity in prostaglandins and thromboxane synthesis: differential activation by Fc- and Cab-dependent bacterial phagocytosis. Prostaglandins 36, 491. Dauber, J.H., Holian, A, Rosemiller, M.E., and Daniele, R.P. (1983) Separation of bronchoalveolar cells from the guinea pig on continuous density gradients of Percoll: morphology and cytochemical properties of fractionated lung macrophages. J. Reticuloendothel. Soc. 33, 119. De Bakker, J.M., De Witt, A.W., Koerten, H.K., Ginsel, L.A. and Daems, W.T. (1985) On the origin of peritoneal resident macrophages. III. EM-immunocytochemical studies on the origin of mouse peritoneal resident macrophages. J. Submicrosc. C~tol. 17, 153. Elias, J.A., Ferro, T.J., Rossman, M.D., Greenberg, J.A., Daniele, R.P., Schreiber, A.D. and Freundlich, B. (1987) Differential prostaglandin production by unfractionated and density-fractionated human monocytes and alveolar macrophages. J. Leukocyte Biol. 42, 114. Ferro, T.J., Kern, J.A., Elias, J.A., Kamoun, M., Daniele, R.P. and Rossman, M.D. (1987) Alveolar macrophages, blood monocytes, and density-fractionate: Alveolar macrophages differ in their ability to promote lymphocyte proliferation to mitogen and antigen. Am. Rev. Respir. Dis. 135, 682. Fishman, M. and Weinberg, D.S. (1979) Functional heterogeneity among peritoneal macrophages II: enzyme content of macrophage subpopulations. Cell. Immunol. 45, 437. Gmelig-Meyling, F. and Waldmann, T.A. (1980) Separation of human blood monocytes and lymphocytes on a continuous Percoll gradient. J. Immunol. Methods 33, 1. Hester, R.B. and Walker, W.S. (1981) Separation of murine mononuclear phagocytes by density gradients of Percoll. In: E.D.O. Adams, P.J. Edelson and H. Koren (Eds.), Methods for Studying Mononuclear Phagocytes. Academic Press, New York, p. 63. Hopper, K.E. and Geczy, C.L. (1980) Characterization of guinea pig macrophages. I. Mobility and maturation of
peritoneal cells following inflammatory stimuli. Cell. Immunol. 56, 400. Kipps, T.J. (1989) The CD5 B cell. Adv. Immunol. 47, 117. Lee, K.C. (1980) On the origin and mode of action of functionally distinct macrophage subpopulation. Mol. Biochem. 30, 39. Leenen, J.M., Slieker, W.A.T., Melis, M. and Van Ewijk, W. (1990) Murine macrophage precursor characterization. I. Production, phenotype and differentiation of macrophage precursor hybrids. Eur. J. Immunol. 20, 15. Meltzer, M.S. (1981) Peritoneal mononuclear phagocytes from small animals. In: E.D.O. Adams, P.J. Edelson and H. Koren (Eds.), Methods for Studying Mononuclear Phagocytes. Academic Press, New York, p. 63. Miller, G.A. and Morahan, P.S. (1982) Functional and biochemical heterogeneity among subpopulations of rat and mouse peritoneal macrophages. J. Reticuloendothel. Soc. 32, 111. Murphy, M.A. and Herscowitz, H.B. (1984) Heterogeneity among alveolar macrophages in humoral and cell-mediated immune responses: separation of functional subpopolations by density gradient centrifugation on Percoll. J. Leukocyte Biol. 35, 39. Oghiso, Y. (1987) Morphologic and functional heterogeneity among rat alveolar macrophage fractions isolated by centrifugation on density gradients. J. Leukocyte Biol. 42, 188. O'Neill, S.J., Hoehm, J.K., Lesperance, E. and Klass, D.J. (1984) Functional heterogeneity of isopycnic fractions of rat alveolar macrohages. Infect. Immun. 46, 282. Pelus, L.M., Broxmeyer, H.E., De Sousa, M. and Moore, M.A.S. (1981) Heterogeneity among resident murine peritoneal macrophages: separation and functional characterization of monocytoid cells producing granulocyte-macrophage colony stimulating factor (GM-CSF) and responding to regulation by lactoferrin. J. Immunol. 126, 1016, 1981. Pertoff, H., Hirtenstein, M. and Kagedal, L. (1979) Cell separations in a new density medium, Percoll. In: E. Reid (Ed.), In Cell Populations, Methodological Surveys. Biochemistry, Vol. 9. Ellis Horwood, Chichester, West Sussex, p. 63. Pertoff, H., Johnsson, A., Warmegard, B. and Seljelid, R. (1980) Separation of human monocytes on density gradients of Percoll. J. Immunol. Methods 33, 221. Plasman, N. and Vray, B. (1993) Mouse peritoneal macrophages: characterization of functional subsets following Percoll density gradients. Res. Immunol. 144, 151. Rasmussen, S.E., Rhodes, J.M., Bennedsen, J. and Larsen, S.O. (1983) Fractionation of untreated and inflammatory murine peritoneal macrophages on discontinuous Percoll density gradients. Acta Pathol. Microbiol. Immunol. Scand. 91, 299. Reppun, T.S., Lin, H.S. and Kuhn, C. (1979) III Isokinetic separation and characterization of mouse pulmonary alveolar colony-forming cells. J. Reticuloendothel. Soc. 25, 379. Sakai, K., Moriya, H., Ueyama, A. and Kishino, Y. (1991) Morphological heterogeneity among fractionated alveolar
B. Pray, N. Plasman /Journal of Immunological Methods 174 (1994) 53-59 macrophages in their release of lysosomal enzymes. Cell. Mol. Biol. 37, 85. Serio, C., Gandour, D.M. and Walker, W.S. (1979) Macrophage functional heterogeneity: Evidence for different antibody- dependent effector cell activities and expression of Fc-receptors among macrophage subpopulations. J. Reticuloendothel. Soc. 25, 197. Shellito, J. and Kaltreider, H.B. (1984) Heterogeneity of immunologic function among subfractions of normal rat alveolar macrophages. Am. Rev. Respir. Dis. 129, 747. Sutton, L.N., Mason, D.Y. and Redman, C.W.G. (1989) Isolation and characterization of human fetal macrophages from placenta. Clin. Exp. Immunol. 78, 437. Thompson, B. and Myrvik, Q.N. (1985) Characterization and
59
maturation of alveolar macrophages procured from BCGinduced pulmonary granulomas. Exp. Lung Res. 9, 237. Tzehoval, E., De Baetselier, P., Feldman, M. and Segal, S. (1981) The peritoneal antigen presenting macrophage: control and immunogenic properties of distinct subpopulations. Eur. J. Immunol. 11,323. Van Furth, R. (1989) Origin and turnover of monocytes and macrophages. In: O.H. Iversen (Ed.), In Cell Kinetics of the Inflammatory Reaction. Current Topics in Pathology. Springer-Verlag, Berlin, p. 125. Wang, S.Y., Mak, K.L., Chen, L.Y., Chou, M.P. and Ho C.K. (1992) Heterogeneity of human blood monocyte: two subpopulations with different sizes, phenotypes and functions. Immunology 77, 298.
JOURNALOF IMMUNOLOGICAL METHODS Journal of ImmunologicalMethods 174 (1994) 53-59
Separation of murine peritoneal macrophages using percoll density gradients B e r n a r d V r a y *, N a t h a l i e P l a s m a n Laboratoire d'Imrnunologie (CP 615), Facult~ de M~decine, Universit~ Libre de Bruxelles, 808 route de Lennik, B-1070 Brussels, Belgium
Abstract
Macrophages harvested from the murine peritoneal cavity are functionally and morphologically heterogeneous. Here, we describe a procedure which permits the determination of specific cell densities using a continuous density gradient of Percoll (analytical step). Subsequently, discontinuous density gradients are used in routine (preparative step) to isolate all the cell subpopulations according to their actual specific density. This procedure has been successfully used for both mouse and rat peritoneal macrophages. Keywords: Murine peritoneal macrophage; Cell density; Cell separation; Continuous density Percoll gradient;
Discontinuous density Percoll gradient
1. Introduction
Due to their central role in the immune system, macrophages are widely used in experimental work. To study the various functions of these cells as well as to analyze their interactions with microorganisms, murine peritoneal macrophages (MPM) are frequently used since they are easy to harvest. However, their high functional and morphological heterogeneity (Bursuker and Goldman, 1983; De Bakker et al., 1985; Van Furth,
Abbreviations: MPM, mouse peritoneal macrophage; HBSS, Hanks' balanced salt solution. * Corresponding author. Tel.: 3225556260; Fax: 322555 6128.
1989; Leenen et al., 1990) and the presence of non-macrophagic cells (Meltzer, 1981; Kipps, 1989) can present problems. Consequently, the fractionation of peritoneal cells into functionally macrophage enriched subpopulations has triggered the development of various procedures based on Stokes' law: in a density gradient, the rate of cell sedimentation in a centrifugal field is zero when the cells encounter a medium of identical density (Pertoff, 1979). Such gradients are prepared either with Percoll (see below) or with Ficoll (Campbell et al., 1980; H o p p e r and Geczy, 1980; Miller and Morahan, 1982) or with bovine serum albumin (Fishman and Weinberg, 1979; Chapes and Tompkins, 1981; Tzehoval et al., 1981). The centrifugation step can also be omitted and cells are separated by velocity sedimenta-
0022-1759/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0022-1759(94)00155-P
54
B. Vray, N. Plasman /Journal of Immunological Methods 174 (1994) 53-59
tion (Reppun et al., 1979; Pelus et al., 1981; Wang et al., 1992). The Percoll density gradient technique has been widely adopted due to its ease of use. In this case, cell separations are based partly on Stokes' law and partly on the cell diameter (Lee, 1980; Coquette, 1989) or the cellular volume (Chandler et al., 1986a). In many studies, macrophages are separated on discontinuous (pre-formed) Percoll gradients. In this case, a relatively large number of density layers is arbitrarily fixed. Consequently, the number of cell subpopulations harvested depends on the number of the Percoll solutions which are overlayered in the tubes (Serio et al., 1979; Hester and Walker, 1981; Rasmussen et al., 1983; Murphy and Herscowitz, 1984; O'Neill et al., 1984; Thompson and Myrvik, 1985; BielefeldtOhmann and Babiuk, 1986; Oghiso, 1987; Ferro et al., 1987; Elias et al., 1987; Sutton et al., 1989; Sakai et al, 1991). On the whole, all these studies show a clear cut difference between cell subpopulations of low and high densities with respect to various functions. An improved cell separation can be obtained using continuous (or self-generated) density gradients of Percoll. A Percoll solution of a given density is centrifuged and high and low densities are distributed around this given density. An optimal cell separation is obtained when all the densities lie along a sigmoidal curve. Consequently, when a heterogeneous cell population is layered on a continuous gradient and centrifuged, numerous cell subpopulations are separated according to their actual specific density. Results obtained with this procedure underline the relationship between cell density and various functions which depend on the maturation stage of the cell (Gmelig-Meyling and Waldmann, 1980; Dauber et al., 1983; Shellito and Kaltreider, 1984). Linear continuous density gradients have also been used for the fractionation of rat alveolar macrophages (Chandler et al., 1986 a and b). Finally, a combination of self-generated and pre-formed gradients readily permits the harvesting of cell subpopulations enriched in macrophages (Coquette et al., 1988; Plasman and Vray, 1993).
The specific cell density of macrophages depends on the strain, the age and the sex of the mice or rats used. For this reason, it is recommended to first determine this specific cell density in an analytical step. This can be done by centrifuging simultaneously a continuous gradient containing coloured beads of well known density together with another gradient of the peritoneal cells to be studied. A direct comparison of the two tubes indicates the specific density of each cell subpopulation. Subsequently, a preparative step using a discontinuous density gradient permits all the cell subpopulations to be separated according to their actual specific density. The procedure that we describe here has been successfully used for both mouse and rat peritoneal macrophages. It could be readily adapted to other types of phagocyte.
2. Materials
Percoll (Pharmacia Fine Chemicals, Sweden) is a polyvinyl-pyrrolidone-coated silica which is supplied as a sterile colloidal suspension with characteristics (density: 1.130 + 0.005 g/ml; pH: 8.9 + 0.3 at 20°C; refraction index: 1.3540 + 0.005 at 20°C) which vary slightly from batch to batch. Other technical data can be found in the handbook Percoll, Methodology and Applications, which is available on request from the manufacturer. Hanks' balanced salt solution without Ca z+ and without Mg 2÷ (pH 7.4, HBSS, Gibco, Grand Island, New York). RPMI 1640 medium containing 10% of fetal calf serum, 100 U / m l penicillin and 100 /~g/ml streptomycin (Gibco). Mice: one mouse yields 2-3 × 106 resident peritoneal cells. 15 male or female mice weighing 25-30 g are required to harvest enough peritoneal cells (30 x 10 6) for one gradient. Cells are pooled and washed in a polypropylene (50 ml volume) conical tube (Becton Dickinson Labware, Falcon no. 2070). Continuous gradient: polystyrene, siliconized tubes forhigh speed centrifugation and adapted to a JA-30 rotor (centrifuge L2 Beckman Instrument Spinco, California, USA).
B. Vray, N. Plasman /Journal of Immunological Methods 174 (1994) 53-59
Discontinuous gradient: polystyrene, siliconized conical tubes (Becton Dickinson Labware, Falcon no. 2095).
3. Methods
55
3.2. Percoll solutions An iso-osmotic solution of Percoll of a given density (1/, working solution) is prepared by mixing sterile sodium chloride solution (1.5 M) and a stock solution of Percoll (1/o) according to the following formula:
3.1. Harvesting of peritoneal cells d - (0.1 × dl0 ) - 0.9 A mouse is killed with ether and pinned, ventral side up, on to a dissecting board under a sterile laminar flow. The abdomen skin is sterilized with ethanol and peeled back. Using a disposable syringe (5 ml) with an 20 gauge needle, peritoneal cells are harvested by two successive washings of the peritoneal cavity with pre-chilled HBSS. The peritoneal cells are gently introduced into a chilled polypropylene 50 ml conical tube (Falcon no. 2070). Then, all the peritoneal cells harvested from the 15 mice are pooled and washed twice in HBSS by centrifugation (300 x g for 10 min at 4°C). Cell pellets are resuspended in RPMI 1640 medium. After counting in a Thoma chamber, cells are adjusted to 1 × 106/ml.
A
1/o = V"
do- 1
where 1/o = volume (ml) of Percoll (stock solution); V = final volume (ml) of the working solution of Percoll; d = final density of the iso-osmotic working solution (g/ml); d o = density of the stock solution of Percoll (g/ml); dl0 = density of a 1.5 M NaCI solution (1.058 g/ml). Example. The density of the stock solution is d o = 1.130 g / m l (indicated on the bottle). To prepare 100 ml (1/) of an iso-osmotic solution of 1.070 g / m l (d), add 49.4 ml of Percoll (stock solution) to 10 ml of NaCI (1.5 M) in a sterile glass bottle and make up to 100 ml with sterile distilled water. Kept sterile, this stock solution
DENSITY (g/ml) 1.140-
I L____~
..- e ~
1.120 ,,./
/"
/ 1.100
./
Or"
O/ /
1.080
%../ II
/0
/
M
61
G;
R1
_.s."
,,,... ! f . , ' ~o
1.060
B1 01
B1
B; B;
1.040
.IO;
01
R;
1.020
V
R1
vR:
1.000 0
51
G; B;
v
3.5
DISTANCE IN CM'S FROM MENISCUS OF GRADIENT
I
II
III
Fig. 1. Density and cell distribution using a continuous Percoll gradient. A: calibration tubes were prepared with Percoll solutions (I: 1.114 g/ml; II: 1.090 g/ml; III: 1.036 g/ml) and mixed with coioured beads of known density. After centrifugation (40000 x g, 30 min, 4°C) in a JA-30 rotor (centrifuge L2 Beckman Instrument Spinco, California, USA), the distances between the meniscus and the coloured bands were plotted. B: distribution of coloured beads in the tubes after centrifugation. From the top to the bottom BI: blue, 1.016 g/ml; O1: orange, 1.033 g/ml; GI: green, 1.048 g/ml; RI: red, 1.062 g/ml; B2: blue, 1.076 g/ml; 02: orange, 1.088 g/ml; G2: green, 1.100 g/ml; R2: red, 1.118 g / m l and V: violet, 1.139 g/ml).
56 can be stored for the dark. The density of by measuring the tometer (Galileo, ture.
B. Vray, N. Plasman/ Journal of lmmunological Methods 174 (1994) 53-59 several days in the cold and in
CELL NUMBER / ml x 106
the Percoll solution is obtained refractive index with a refracMilan, Italy) at room tempera-
5-
lo
4-
3-
3.3. Preparation of an optimal continuous gradient profile
!
DENSITY ( g / m l )
1,139
1.118 1,100 1.088 1.076
2 1.062
The generation of a continuous gradient profile depends on various physical parameters. Before using the peritoneal cells, it is recommended to first test that the density gradient exhibits a sigmoidal profile. For this, a Percoll solution (1.090 g / m l , 40 ml) is prepared and chilled. To measure the distance between the meniscus and various coloured bands, an adhesive millimeter scale is attached to a calibration tube and 10/xl aliquots of the different coloured beads are added to 20 ml of the Percoll solution. The tube is then centrifuged (40000 x g, 30 min, 4°C) in a JA-30 rotor (centrifuge L2 Beckman Instrument Spinco, California, USA) and the density profile of the gradient is determined by plotting the distance between the meniscus and the different layers of coloured beads. As shown in Fig. 1 a, a sigmoidal profile is obtained with a density of 1.090 g / m l , ensuring an optimal cell separation: all the densities are distributed all along the top and the bottom of the tube (Fig. lb). By contrast, two other Percoll densities (1.036 and 1.114 g / m l ) give completely different profiles. 3.4. Determination o f specific cell densities with a continuous density gradient A pellet of peritoneal cells (30 x 106/ml) is mixed with 20 ml of Percoll solution (1.090 g / m l ) in a siliconized sterile polystyrene tube (experimental tube). A calibration tube prepared as described above is prepared and the two tubes are centrifuged together (40000 x g, 30 min, 4°C). Then, fractions of 250/zl are collected from the experimental tube, using an 18-gauge spinal needle connected to a peristaltic pump (model 2120 - LKB Instruments, Bromma, Sweden). Cells are washed twice in cold HBSS, resuspended in 250
1.048
1 234 0
•
1.033
7 i
I
1.016
I
2o
10
VOLUME ( ml )
I
0
315 DISTANCE IN CM'S FROM
MENISCUS OF GRADIENT
Fig. 2. Mouse peritoneal cells were harvested from male BALB/c mice. They were layered on the top of a continuous density gradient of Percoll (density 1.090 g/ml) and centrifuged. The figure illustrates the sigmoidal profile of the densities associated with the 12 subpopulations obtained after centrifugation.
/xl HBSS and counted. In our experimental model, 12 cell subsets are identified with male B A L B / c mice (Fig. 2) and 14 subsets with female mice. 3.5. Discontinuous density gradient To harvest large amounts of fractionated peritoneal cells, discontinuous gradients are prepared as above. 1 ml of a Percoll solution (1.117 g / m l ) is first poured into siliconized polystyrene tubes. Then, 1 ml of each specific cell density Percoll solution is layered from the heaviest at the bottom to the lighest at the top. Finally, 1 ml of Percoll (1.010 g / m l ) containing 3 × 107 cells/ml is layered on to the top of the gradient. The discontinuous gradient is centrifuged (400 X g, 4°C, 30 min) in a swinging bucket rotor (centrifuge J O U A N CR411, Saint Nazaire, France). Then, the various cell subsets are collected by suction with a siliconized sterile Pasteur pipette, washed twice in cold HBSS and resuspended to the appropriate cell concentration and can be used for microscopic observations, enzymatic, flow-cytometric and phagocytic
B. Vray, N. Plasman/Journal of Immunological Methods 174 (1994) 53-59
assays. The viability of cells is estimated by the trypan blue dye exclusion test.
4. Critical comments
Percoll appears to be the medium of choice for cell separation. Indeed, our results indicate a high percentage of cell viability together with the preservation of critical functions such as bacterial phagocytosis or eicosanoid synthesis (Coquette et al., 1988; Plasman and Vray, 1993). They also confirm previous work indicating that other cell functions are not affected (Serio et al., 1979; Gmelig-Meyleng and Waldmann, 1980; Pertoff et al., 1980). To inhibit spontaneous cell agregation, separations have to be performed with chilled materials and medium lacking Ca a+ and Mg 2+. Cell concentration is also critical and depends on the surface at the interface between Percoll layers. Thus, the choice of the tubes and their internal diameter is limiting for the cell suspension layered on the top of the gradient. Murine peritoneal cells include mainly macrophages (60-70%) with many lymphocytes (3040%) recovered in low density fractions. However, as a consequence of their large spectrum of densities, some lymphocytes are also present in macrophage enriched fractions. Most of these "contaminating" lymphocytes can be discarded by plating subsets for 2 h in culture dishes and allowing the macrophages to adhere. Subsequent washing with RPMI 1640 will remove most of the lymphocytes. Other non-macrophagic cells (neutrophils, erythrocytes and mast cells) are also encountered but at a very low percentage of the total population. They are found in high density fractions and at the bottom of the tube (Plasman and Vray, 1993). Fractionation of MPM into subpopulations depends on their specific cell density and cell diameter. These two parameters seem to be related to the maturation of the cell which does not necessarily correlate with physiological function. However, we have observed that the rate of bacterial phagocytosis, both antibody and complement mediated, and eicosanoid synthesis are related to macrophage density (Coquette et al., 1988). This
57
is also the case for rat alveolar macrophages (Chandler et al., 1986 a and b). Cell specific density depends on various parameters (e.g. the species, strain, age and sex of the animals used as well as the state of husbandry). Others parameters (choice of tubes, rotors and centrifuges) also influence the acccuracy of cell separations. Thus, it must be stressed that in order to obtain the continuous density gradient of Percoll permitting the best cell separations, it is necessary to test different densities together with centrifugation procedures of differing duration and speed.
5. Conclusion
Macrophages harvested from different anatomical sites are heterogeneous and this has hampered the analysis of many of their basic functions. Percoll density gradients offer the opportunity to eliminate most of the non-macrophagic cells and distribute macrophages of differing specific density according to their maturation stage and cell size.
Acknowledgments
The authors thank Mr. V. Vercruysse for excellent technical assistance, Mr. I. Mazza and Mr. N. Cardon for help in preparing the manuscript. This work was supported by grant "Action de Recherche Concert6e", U.L.B., 1991 and the Foundation M. M6rieux.
References
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