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Isolation of rat peritoneal mast cells by centrifugation on density gradients of Percoll
Journal of ImmunologicaI Methods, 39 (1980) 135--145
135
© Elsevier/North-Holland Biomedical Press
ISOLATION OF RAT PERITONEAL MAST CELLS BY CENTRIFUGATION ON DENSITY GRADIENTS OF PERCOLL
LENNART ENERBACK * and IRENE SVENSSON
Department of Pathology H, University of Link~ping, S-581 85 Link6ping, and Department of Pathology II, University of G6teborg, S-413 45 G6teborg, Sweden (Received 18 March 1980, accepted 9 July 1980)
Rat mast cells from peritoneal washings were purified by centrifugation on a medium containing silica particles coated with PVP (Percoll). By isopycnic centrifugation on continuous Percoll gradients, the buoyant density of mast cells ranged from 1.09 to 1.17 and was consistently lower in mast cells from younger rats than in mast cells from older rats. In suspensions uncontaminated with blood, the mast cells could be isolated by a simple centrifugation procedure based on density differences (step I). Contaminating red blood cells were removed by a second short centrifugation of velocity gradient type, using a linear Percoll gradient of low density. The mast cell purity was 91--98% and the recovery 63--100%. Purified mast cells reacted normally in a viability test employing fluorescein diacetate, retained amines and heparin, and reacted normally with amine release following incubation with Polymyxin B or antigen (immunised rats). Electron microscopy revealed excellent ultrastructural preservation of the mast cells and infrequent uptake Of Percoll particles by endocytosis.
INTRODUCTION
Peritoneal cell suspensions have been much used in studies on the immunobiology of mast cells. By washing out the peritoneal cavity, single cell suspensions containing 2--5% mast cells may easily be obtained. However, in many studies there is a need to obtain the cells in pure form. A number of methods have therefore been devised for isolation of mast cells from peritoneal cell suspensions. Most of these are based on density gradient centrifugation. The earliest development in this field employed sucrose-gelatin or sucrose (Padawer and Gordon, 1955; Glick et al., 1956). However, these methods were unsatisfactory because of damage to the mast cells and loss of significant quantities of histamine during the isolation a n d subsequent incubation procedures (Uvn~is and Thon, 1961; Johnson and Moran, 1966). Density gradient centrifugation on Ficoll (Uvn~ and Thon, 1961; Bach et al., 1971; Cooper and Stanworth, 1976) or albumin (Keller, 1966; Lagunoff, * T o w h o m correspondence should be addressed. Present address: Department of Pathology II, University of GSteborg, S-413 45 GSteborg, Sweden.
136
1975; Sullivan et al., 1975) has been preferred by many investigators. These methods are not without problems, however. The methods employing Ficoll are time consuming and the medium may be difficult to remove from the isolated cells. It has also been claimed that cells isolated on Ficoll have low viability and lose some of their reactivity when exposed to antigens following immunisation (Sullivan et al., 1975; Karlsson, 1978). We have obtained mast cell suspensions with high purity using centrifugation on albumin (Lagunoff, 1975) but there was some loss of histamine, and difficulty in obtaining a satisfactory yield of mast cells from the peritoneal cell suspensions (Enerb~ick and Wingren, 1980a). A new medium for gradient centrifugation containing silica particles coated with thin layers of polyvinyl pyrrolidone (PVP) has recently been described (Pertoft and Laurent, 1977; Pertoft et al., 1978). This colloid (Percoll) has many advantages for cell separation purposes. It has low osmolality and viscosity and can be mixed with various physiological solutions. In addition, density gradients are self-generated during high speed centrifugation. Previous studies have indicated that the PVP coated colloid has a low toxicity (Pertoft et al., 1977). In this paper, a simple and rapid method for isolation of peritoneal mast cells employing Percoll is described. The method gives mast cell suspensions with high yield and purity. The mast cells retain their heparin and 5-hydroxytryptamine content during purification and react in a normal way to stimulation of secretion by antigen or polymyxin B. In a viability test with fluorescein diacetate the purified mast cells reacted like the mast cells in crude suspensions and all retained fluorescent material. MATERIALS AND METHODS
Male Sprague--Dawley rats (obtained from Anticimex AB, Stockholm, Sweden) and Hooded Lister rats, obtained from a colony bred at Biomedical Center, University of Uppsala, were used. Crude peritoneal mast cell suspensions containing about 5% mast cells were obtained by washing out the peritoneal cavity with Hank's solution containing 0.1% bovine serum albumin (HA), as described previously (Enerb~ick, 1974).
Reagents Percoll is a product of Pharmacia Fine Chemicals (Uppsala, Sweden). Isotonic (approximately 300 mOsm/kg H20) Percoll solutions were prepared by dissolving 9 parts of Percoll with 1 part of 10-fold concentrated HA. Coloured beads obtained from Pharmacia Fine Chemicals covering the buoyant density range 1.016--1.178 g/ml were used for calibration of Percoll gradients and for checking equilibrium conditions (see below). Polymyxin B, bovine serum albumin and egg albumin were obtained from Sigma (KemilaProdukter AB, Stockholm).
137
Centrifugation technique A Sorvall RC 2-B superspeed centrifuge was used. All centrifugations were done at 4 ° C. Continuous density gradients of Percoll were generated by centrifugation at 30,000 X g for 100 min in a Sorval SS-34 fixed angle rotor. Gradients ranging from a density of 1.04 to 1.18 were obtained by this procedure. Density of solutions was measured on a density column prepared b y mixing carbon tetrachloride and ligroin according to the principle described by Jacobsen and LinderstrSm-Lang (1940), or b y weighing.
Cytofluorometric quantitations 5 - H y d r o x y t r y p t a m i n e (5-HT) and heparin were measured by previously described cytofluorometric methods (Enerb~ick, 1974; Enerb~ick et al., 1977). Measurements on individual mast cells in population samples of 200 cells were made in a microscope fluorometer as described previously (Mellblom and Enerb~ick, 1979). 5-HT is bound at the same storage site in the mast cell granules as histamine and may be used as a probe for histamine in studies on amine release (Enerb~ick and Wingren, 1980b).
Quantitation of amine re;ease from mast cells Rats were injected subcutaneously with 100 pCi [3H] 5-HT/100 g b o d y weight together with 25 mg unlabelled 5-HT as carrier. The rats were killed 24 h later and the mast cells used for release studies. Rats used for immunological challenge were immunised with egg albumin according to a technique described by Karlsson (1978). For the amine release studies purified mast cells were incubated at 37°C for 5 min in HA with the addition of p o l y m y x i n B or egg albumin (immunised rats). The reaction was stopped b y transfe~ing the test tubes to an ~ce bath. The tubes were centrifuged at 400 X g for 10 min. Pellets and supernatants were extracted with perchloric acid containing 0.04% EDTA and 0.02% ascorbic acid after freezing and thawing three times. The extracts were neutralised by t h e addition of 5 M K2CO3 and purified b y ion exchange chromatography on a weak cationic exchange resin (And~n and Magnusson, 1967). Activity of cell pellets and supernatants, before and after purification b y column chromatography, was measured in a Packard TriCarb liquid scintillation spectrometer. Percentage amine release was calculated as the fraction of 5-HT remaining in the cell pellets (purified or unpurified extracts) or by the fraction of the total activity found in the supernatant (unpurified extracts only). The t w o types of calculations gave comparable results.
Morphological studies Crude peritoneal cells and isolated mast cells were fixed in 2.5% glutarald e h y d e (0.1 M cacodylate buffer, pH 7.4) for 15 min at 0°C followed by 45
138
min at room temperature, washed in 0.1 M cacodylate buffer, postfixed in 1% osmium tetroxide for 1 h at 0°C, washed in cacodylate buffer and pelleted in agar. The agar pellets were dried, dehydrated and e m b e d d e d in Epon. Thin sections were stained with uranyl acetate and lead nitrate. For light microscopy, 0.5--1 pm thick sections were stained with toluidine blue (1% in 2.5% sodium carbonate, pH 11.1 for 30--120 min). RESULTS
Equilibrium density centrifugation In order to establish a basis for the centrifugation technique, the b u o y a n t density of mast cells was studied in relation to other cells in the peritoneal suspensions by gradient centrifugation on Percoll under equilibrium conditions. Cell suspensions from one rat containing a b o u t 106 mast cells and 20 X 106 total cells suspended in HA were layered on Percoll gradients prepared by high speed centrifugation. A Sorvall HB4 swing-out rotor was used. Centrifugation was performed at 500 × g for 30 min. Equilibrium conditions were checked conveniently with beaded density markers. Good separation of the mast cells from other nucleated cells was easily obtained. Red blood cells, on the other hand, often sedimented with the mast cells. A constant finding was that mast cells from y o u n g animals had consistently lower buoyo
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Fig. 1. Equilibrium density gradient centrifugation on a continuous Percoll gradient generated by high speed centrifugation. Cell suspension from a young rat, weighing 250 g. Gradient ($), mast cells (D), other nucleated cells (x), red blood cells (o). Fig. 2. Equilibrium density gradient centrifugation as in Fig. 1. Cell suspension from an old rat, weighing 500 g. Symbols as in Fig. 1.
139
ant density than mast cells from old rats. The results of t w o typical experiments are shown in Figs. 1 (young rat) and 2 (old rat). The mast cells had a density between 1.09 and 1.15 in the y o u n g rat and between 1.11 and 1.17 in the old rat. In both instances the red blood cells showed a narrow density range of 1.10--1.12 with a peak at 1.11. The results in Figs. t and 2 demonstrate clearly that mast cells m a y be readily separated from nucleated cells b y centrifugation based on density alone. Especially in y o u n g rats, however, good separation from red blood cells cannot be expected by a centrifugation technique based u p o n density only.
Purification o f peritoneal mast cells: step I Based on the above, the following simple technique was devised for purification of mast cells from peritoneal cell suspensions uncontaminated with blood. An aliquot of 0.75 ml of the cell suspensions in HA was added to 3.5 ml Percoll isotonic solution resulting in a final density of 1.110. The solution was mixed thoroughly and allowed to settle. Then, 0.5 ml 0.1% HA was layered on t o p of the solution and the sample was centrifuged at 125 X g for 15 min. The t w o uppermost ml were removed together with 0.5 ml HA used for washing the wall of the test tube. The remaining volume containing the mast cells was washed twice in 0.1% HA (centrifugation at 300 X g and for 5 min). The results of the procedure are shown in Table 1. Four animals in each of three age groups were used because of the marked influence of age on the density of the mast cells. The Table shows satisfactory purity and recovery. The suspensions contained few red blood cells. The recovery of mast cells was dependent on the age of the animals b u t was also satisfactory for the youngest age group (72%).
TABLE 1 Mast cell p u r i f i c a t i o n b y d e n s i t y g r a d i e n t c e n t r i f u g a t i o n (step I). R e s u l t s are m e a n s + S.E.M. Age (days) a
45 62 102
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200-+3 311-+4 3 9 2 -+ 4
Cell c o n c e n t r a t i o n (%) MC b
R B c.
OC d
Mast cell yield (%)
91+1 92-+1 9 3 -+ 1
7+1 4-+1 4 -+ 1
3-+0 5-+1 5 -+ 0
72-+2 85-+4 100 -+ 6
a T h r e e e x p e r i m e n t s o n 4 d i f f e r e n t rats in e a c h group. b Mast cells, c r e d b l o o d cells, d o t h e r cells.
140
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Fig. 3. Velocity gradient centrifugat!on (40 x g, 5 min) with added red blood cells. Shaded columns are mast cells and open cqlumns red blood cells.
Purification of peritoneal mast cells: step H The previously described results (Table 1) showed that isolation of mast cells with high yield and purity could be achieved by a simple centrifugation technique based on density, provided that the cell suspensions were not contaminated with blood. The 12 consecutive experiments illustrated in Table 1 showed that contamination with blood cells could be avoided to a large extent. However, for cases when such contamination occurred, we devised a method for removing red blood cells. Since a separation based on density
TABLE 2
Mast cell purification by combined density (step I) and velocity gradient centrifugation (step II). Cell suspensions from 6 rats aged 57 -+ 1 days. Red blood cells added to cell suspensions. Results are means + S.E.M. Cell c o n c e n t r a t i o n (%)
Step I
Step II
Mast cells Red blood cells Other cells
65 -+ 2 33 -+ 2 2 -+ 0
98+1
Mast cell yield Total mast cell yield
78 + 2
1_+0 1_+0 82-+7 63+4
141
was n o t feasible, a velocity gradient type of centrifugation procedure was selected. A continuous linear Percoll gradient with a density of 1.020-1.030 was prepared with a peristaltic pump. Mast cell suspensions containing red blood cells were layered on t o p of the gradient and centrifuged for 5 min at 40 X g (HB 4 swing-out rotor). The result of an experiment employing analysis in 3 ml fractions is shown in Fig. 3. It is evident that mast cells could be separated from the red blood cells with this t y p e of centrifugation procedure. As a standard technique we used a gradient with a volume of 36 ml with a density ranging from 1.02 to 1.03 on a cushion of 2 ml Percoll, density 1.12. Two ml of a cell suspension was layered on t o p of this gradient. After centrifugation (40 X g for 5 min), 25 ml of the suspension (containing the red blood cells) was removed by pumping from the top of the tubes. The remaining solution was washed twice in 0.1% HA and resuspended in a desired volume of HA. Results of the complete separation procedure performed on 6 young rats to the peritoneal cell suspensions of which red blood cells had been added is shown in Table 2. These results show clearly that blood cells were removed b y the second step of the procedure. The total mast cell yield was satisfactory. Heparin and 5-HT content o f mast cells
In order to check possible selection of mast cells during the centrifugation procedure, or leakage of amines, cytofluorometric quantitations of 5-HT and heparin were performed on peritoneal mast cells before and after purification. For this experiment the complete purification procedure was used. We analysed mast cell suspensions from 3 rats. The results in Table 3 demonstrate that both the 5-HT and heparin content were unaffected by the purification procedure. Mast cell secretion studies
Mast cell secretion was studied in purified mast cell suspensions from Sprague--Dawley rats using p o l y m y x i n B as a secretagogue. The results shown in Fig. 4 demonstrate that the cells reacted p r o m p t l y with amine secretion. The reactivity, in terms of histamine release was of similar magnitude to that which we previously found in mast cells of crude peritoneal cell suspensions (Gustafsson and Enerb~ick, 1980). IgE~lependent mast cell secretion was studied in h o o d e d Lister rats immunised with egg albumin according to a technique optimised for production of IgE antibodies (Karlsson, 1978). The results of two experiments involving secretion stimulation by challenge with the antigen are shown in Fig. 5. The resulting maximum histamine release was 30--50% which indicates that the reactivity of the mast cell towards immunological challenge was n o t reduced b y the purification procedure. Spontaneous histamine release was low in all three experiments (3--7%).
142 TABLE 3 5 - H y d r o x y t r y p t a m i n e and h e p a r i n c o n t e n t o f m a s t cell p o p u l a t i o n s b e f o r e and a f t e r purif i c a t i o n ( c o m b i n e d p r o c e d u r e , step I + II). Cell s u p e n s i o n s a
5-Hydroxytryptamine b
Heparin b
1 Crude Pure 2 Crude Pure 3 Crude Pure
105 108 131 137 106 116
171 175 164 164 158 163
-+ 4 -+ 3 -+ 5 + 4 -+ 3 + 3
-+ 3 -+ 3 -+ 3 -+ 3 + 4 + 4
a Crude p e r i t o n e a l cell s u s p e n s i o n s c o n t a i n i n g 3--5% mast cells and purified m a s t cell s u s p e n s i o n s c o n t a i n i n g 9 1 - - 9 6 % m a s t cells f r o m each o f 3 d i f f e r e n t rats. b Measured b y m i c r o s c o p e f l u o r o m e t r y in p o p u l a t i o n samples o f 200 m a s t cells. Populat i o n m e a n -+ S.E.M,
Viability test using fluorescein diacetate (FDA ) Crude or purified mast cell suspensions were incubated in solutions of 2 pM FDA in HA. Fluorescence developed rapidly in the mast cells and after 10 min incubation at room temperature, more than 98% of the mast cells in crude and purified mast cell suspensions showed a strong fluorescence.
Morphology o f isolated mast cells Morphology as determined by both light microscopy and electron microscopy was extremely well preserved in mast cells purified by the centrifuga/ P.................
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Fig. 4. A m i n e release f r o m purified m a s t cells i n c u b a t e d w i t h p o l y m y x i n B at 36°C. A m i n e release was assayed in t e r m s o f release o f radiolabelled 5-HT in m a s t cell suspensions f r o m rats i n j e c t e d 24 h prior t o sacrifice w i t h 100 pCi 3H-labelled 5-HT s u b c u t a n e ously.
Fig. 5. IgE-dependent mast cell secretion by challenge with egg albumin in rats immunised for IgE production 2 weeks before sacrifice.T w o separate experiments.
143 iii
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Fig. 6. Electron micrographs of mast cells isolated by gradient centrifugation on Percoll. Endocytotic vesicles containing Percoll particles indicated by arrows. ] ]= 1 pm (upper two figures) and 0.36 pm (lower two figures).
tion procedure. However, by electron microscopy purified mast cells were occasionally found to contain small electron-dense particles enclosed within endocytotic vesicles in the peripheral parts of the cells (Fig. 6). These particles were similar in size to Percoll particles, 10--20 nm (Pertoft et al., 1978). DISCUSSION
The results reported in this paper show that mast cells can be isolated with high yield and purity by centrifugation on gradients of Percoll. This medium, consisting of PVP-coated silica particles has properties which makes it highly
144
desirable for cell separation purposes (Pertoft et al., 1978). Of special importance is its extremely low osmotic pressure, which makes it possible to prepare gradients with high density in various physiological media, and its property of forming continuous gradients during high speed centrifugation. Such continuous Percoll gradients were used with isopycnic centrifugation under equilibrium conditions to determine the b u o y a n t density of the mast cells. We found that mast cells had a higher density than other nucleated cells in crude peritoneal cell suspensions. Moreover, mast cells from young rats had consistently lower density than mast cells from older animals. We found density values for mast cells ranging from 1.09 to 1.17 g/ml. The b u o y a n t density of mast cells has previously been studied by Pretlow and Cassady (1970) w h o reported density ranging from 1.08 to 1.29 in another strain of rats. We have previously demonstrated that dry mass, protein, heparin and amine content all increase in peritoneal mast cells as a function of age in growing rats (Enerb~ick and Mellblom, 1978; Mellblom and Enerb~ick, 1979). Since mast cells are elements with long lives, such differences between y o u n g and old rats are at least to some extent related to aging of the mast cells (Mellblom, 1979). Mast cells thus had a higher b u o y a n t density than other nucleated cells in the crude peritoneal cell suspensions. The red blood cells showed densities between 1.10 and 1.12, overlapping the low density ranges of mast cells. In y o u n g rats there was also some overlap between mast cells and other nucleated cells in the peritoneal cavity. In spite of this, peritoneal mast cells could be isolated with high yield and purity from the crude peritoneal cell suspensions with a simple centrifugation procedure based on density differences. The procedure also gave satisfactory results in cell suspensions from young animals. For the infrequent occasions when peritoneal cell suspensions are contaminated with blood, red blood cells can be eliminated by a subsequent brief centrifugation of velocity gradient type. The two cell types separate owing to differences in cell size rather than differences in density. The morphology of mast cells was excellently preserved after centrifugation on Percoll, judged both by light microscopy and ultrastructural criteria. However, limited uptake of Percoll particles appeared to take place by an endocytotic mechanism. Since this is most probably a temperature dependent p h e n o m e n o n it could possibly have been avoided by lowering the temperature. However, mast cells isolated on Percoll were well preserved with respect to chemical composition, viability and secretory capacity, both after stimulation with polymyxin and with antigen. The cytofluorometric measurements on individual mast cells of crude and purified suspensions further demonstrated that amine leakage, which appears to occur frequently with other isolation procedures, did not occur with the present method. Heparin, which is a matrix c o m p o n e n t of mast cell granules, was likewise not affected b y the purification procedure, indicating that a loss of mast cell granules did n o t occur, and also, something which is most important, that selection of mast cells with respect to heparin content or mass (see Mellblom, 1980) did
145
n o t occur during the centrifugation procedure. Another indication of good preservation of the mast cells was the finding of very low spontaneous amine release in the secretion studies of isolated mast cells. The simple one step centrifugation procedure for isolation of mast cells appears to be ideally suited for preparation of cells for secretion studies in vitro. ACKNOWLEDGEMENT
This work was supported b y grants from the Swedish Medical Research Council, Project No. 2235. REFERENCES And~n, N.E. and T. Magnusson, 1967, Acta Physiol. Scand. 69, 87. Bach, M.K., K.J. Block and K.F. Austen, 1971, J. Exp. Med. 133,752. Cooper, P.H. and D.R. Stanworth, 1976, in: Methods in Cell Biology, Vol. 14, ed. P.M. Prescott (Academic Press, New York) p. 365. Enerb~ick, L., 1974, Histochemistry 4 2 , 3 0 1 . Enerb~/ck, L. and L. Mellblom, 1978, Cell Tiss. Res. 187,367. Enerb~/ck, L. and U. Wingren, 1980a, Histochemistry 6 6 , 1 1 3 . Enerb~/ck, L. and U. Wingren, 1980b, J. Histochem. Cytochem., in press. Enerb~/ck, L., B. Gustafsson and L. Mellblom, 1977, J. Histochem. Cytochem. 27, 32. Glick, D., S.J. Bonting and D. BenBoer, 1956, Proc. Soc. Exp. Biol. Med. 92,357. Gustafsson, B. and L. Enerb~/ck, 1980, Exp. Cell Biol. 48, 15. Jacobsen, C.F. and K. LindenstrSm-Lang, 1940, Acta Physiol. Scand. 1,149. Johnson, A.R. and N.C. Moran, 1966, Proc. Soc. Exp. Biol. Med. 1 2 3 , 8 8 6 . Karlsson, T., 1978, in: Immunoglobulin E and the Mast Cell, Doctoral Thesis (Uppsala University, Uppsala) p. 26. Keller, R., 1966, in: Tissue Mast Cells in Immune Reactions (American Elsevier, New York) p. 50. Lagunoff, D., 1975, in: Techniques of Biochemical and Biophysical Morphology, Vol. 2, eds. D. Glick and R.M. Rosenbaum (Wiley-Interscience, New York) p. 2 8 3 . . Mellblom, L., 1979, Growth Related Changes of Connective Tissue Mast Cells, LinkSping University medical dissertation No. 82. Mellblom, L., 1980, Cell Tiss. Res., in press. Mellblom, L. and L. Enerb~/ck, 1979, Histochemistry 6 3 , 1 2 9 . Padawer, J. and A.S. Gordon, 1955, Proc. Soc. Exp. Biol. Med. 88, 29. Pertoft, H. and T.C. Laurent, 1977, in: Methods of Cell Separation, Vol. 1, ed. N. Catsimpoulas (Plenum, New York) p. 25. Pertoft, H., K. Rubin, L. Kjell~n, T.C. Laurent and B. Klingeborn, 1977, Exp. Cell Res. 110,449. Pertoft, H., T.C. Laurent, T. L~i~isand L. K~igedal, 1978, Anal. Biochem. 88, 271. Pretlow, T.G. and J.M. Cassady, 1970, Am. J. Pathol. 6 1 , 3 2 3 . Sullivan, T.J., K.L. Parker, W. Stenson and C.W. Parker, 1975, J. Immunol. 114, 1473. U v n ~ , B. and I.-L. Thon, 1961, Exp. Cell Res. 23, 45.
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© Elsevier/North-Holland Biomedical Press
ISOLATION OF RAT PERITONEAL MAST CELLS BY CENTRIFUGATION ON DENSITY GRADIENTS OF PERCOLL
LENNART ENERBACK * and IRENE SVENSSON
Department of Pathology H, University of Link~ping, S-581 85 Link6ping, and Department of Pathology II, University of G6teborg, S-413 45 G6teborg, Sweden (Received 18 March 1980, accepted 9 July 1980)
Rat mast cells from peritoneal washings were purified by centrifugation on a medium containing silica particles coated with PVP (Percoll). By isopycnic centrifugation on continuous Percoll gradients, the buoyant density of mast cells ranged from 1.09 to 1.17 and was consistently lower in mast cells from younger rats than in mast cells from older rats. In suspensions uncontaminated with blood, the mast cells could be isolated by a simple centrifugation procedure based on density differences (step I). Contaminating red blood cells were removed by a second short centrifugation of velocity gradient type, using a linear Percoll gradient of low density. The mast cell purity was 91--98% and the recovery 63--100%. Purified mast cells reacted normally in a viability test employing fluorescein diacetate, retained amines and heparin, and reacted normally with amine release following incubation with Polymyxin B or antigen (immunised rats). Electron microscopy revealed excellent ultrastructural preservation of the mast cells and infrequent uptake Of Percoll particles by endocytosis.
INTRODUCTION
Peritoneal cell suspensions have been much used in studies on the immunobiology of mast cells. By washing out the peritoneal cavity, single cell suspensions containing 2--5% mast cells may easily be obtained. However, in many studies there is a need to obtain the cells in pure form. A number of methods have therefore been devised for isolation of mast cells from peritoneal cell suspensions. Most of these are based on density gradient centrifugation. The earliest development in this field employed sucrose-gelatin or sucrose (Padawer and Gordon, 1955; Glick et al., 1956). However, these methods were unsatisfactory because of damage to the mast cells and loss of significant quantities of histamine during the isolation a n d subsequent incubation procedures (Uvn~is and Thon, 1961; Johnson and Moran, 1966). Density gradient centrifugation on Ficoll (Uvn~ and Thon, 1961; Bach et al., 1971; Cooper and Stanworth, 1976) or albumin (Keller, 1966; Lagunoff, * T o w h o m correspondence should be addressed. Present address: Department of Pathology II, University of GSteborg, S-413 45 GSteborg, Sweden.
136
1975; Sullivan et al., 1975) has been preferred by many investigators. These methods are not without problems, however. The methods employing Ficoll are time consuming and the medium may be difficult to remove from the isolated cells. It has also been claimed that cells isolated on Ficoll have low viability and lose some of their reactivity when exposed to antigens following immunisation (Sullivan et al., 1975; Karlsson, 1978). We have obtained mast cell suspensions with high purity using centrifugation on albumin (Lagunoff, 1975) but there was some loss of histamine, and difficulty in obtaining a satisfactory yield of mast cells from the peritoneal cell suspensions (Enerb~ick and Wingren, 1980a). A new medium for gradient centrifugation containing silica particles coated with thin layers of polyvinyl pyrrolidone (PVP) has recently been described (Pertoft and Laurent, 1977; Pertoft et al., 1978). This colloid (Percoll) has many advantages for cell separation purposes. It has low osmolality and viscosity and can be mixed with various physiological solutions. In addition, density gradients are self-generated during high speed centrifugation. Previous studies have indicated that the PVP coated colloid has a low toxicity (Pertoft et al., 1977). In this paper, a simple and rapid method for isolation of peritoneal mast cells employing Percoll is described. The method gives mast cell suspensions with high yield and purity. The mast cells retain their heparin and 5-hydroxytryptamine content during purification and react in a normal way to stimulation of secretion by antigen or polymyxin B. In a viability test with fluorescein diacetate the purified mast cells reacted like the mast cells in crude suspensions and all retained fluorescent material. MATERIALS AND METHODS
Male Sprague--Dawley rats (obtained from Anticimex AB, Stockholm, Sweden) and Hooded Lister rats, obtained from a colony bred at Biomedical Center, University of Uppsala, were used. Crude peritoneal mast cell suspensions containing about 5% mast cells were obtained by washing out the peritoneal cavity with Hank's solution containing 0.1% bovine serum albumin (HA), as described previously (Enerb~ick, 1974).
Reagents Percoll is a product of Pharmacia Fine Chemicals (Uppsala, Sweden). Isotonic (approximately 300 mOsm/kg H20) Percoll solutions were prepared by dissolving 9 parts of Percoll with 1 part of 10-fold concentrated HA. Coloured beads obtained from Pharmacia Fine Chemicals covering the buoyant density range 1.016--1.178 g/ml were used for calibration of Percoll gradients and for checking equilibrium conditions (see below). Polymyxin B, bovine serum albumin and egg albumin were obtained from Sigma (KemilaProdukter AB, Stockholm).
137
Centrifugation technique A Sorvall RC 2-B superspeed centrifuge was used. All centrifugations were done at 4 ° C. Continuous density gradients of Percoll were generated by centrifugation at 30,000 X g for 100 min in a Sorval SS-34 fixed angle rotor. Gradients ranging from a density of 1.04 to 1.18 were obtained by this procedure. Density of solutions was measured on a density column prepared b y mixing carbon tetrachloride and ligroin according to the principle described by Jacobsen and LinderstrSm-Lang (1940), or b y weighing.
Cytofluorometric quantitations 5 - H y d r o x y t r y p t a m i n e (5-HT) and heparin were measured by previously described cytofluorometric methods (Enerb~ick, 1974; Enerb~ick et al., 1977). Measurements on individual mast cells in population samples of 200 cells were made in a microscope fluorometer as described previously (Mellblom and Enerb~ick, 1979). 5-HT is bound at the same storage site in the mast cell granules as histamine and may be used as a probe for histamine in studies on amine release (Enerb~ick and Wingren, 1980b).
Quantitation of amine re;ease from mast cells Rats were injected subcutaneously with 100 pCi [3H] 5-HT/100 g b o d y weight together with 25 mg unlabelled 5-HT as carrier. The rats were killed 24 h later and the mast cells used for release studies. Rats used for immunological challenge were immunised with egg albumin according to a technique described by Karlsson (1978). For the amine release studies purified mast cells were incubated at 37°C for 5 min in HA with the addition of p o l y m y x i n B or egg albumin (immunised rats). The reaction was stopped b y transfe~ing the test tubes to an ~ce bath. The tubes were centrifuged at 400 X g for 10 min. Pellets and supernatants were extracted with perchloric acid containing 0.04% EDTA and 0.02% ascorbic acid after freezing and thawing three times. The extracts were neutralised by t h e addition of 5 M K2CO3 and purified b y ion exchange chromatography on a weak cationic exchange resin (And~n and Magnusson, 1967). Activity of cell pellets and supernatants, before and after purification b y column chromatography, was measured in a Packard TriCarb liquid scintillation spectrometer. Percentage amine release was calculated as the fraction of 5-HT remaining in the cell pellets (purified or unpurified extracts) or by the fraction of the total activity found in the supernatant (unpurified extracts only). The t w o types of calculations gave comparable results.
Morphological studies Crude peritoneal cells and isolated mast cells were fixed in 2.5% glutarald e h y d e (0.1 M cacodylate buffer, pH 7.4) for 15 min at 0°C followed by 45
138
min at room temperature, washed in 0.1 M cacodylate buffer, postfixed in 1% osmium tetroxide for 1 h at 0°C, washed in cacodylate buffer and pelleted in agar. The agar pellets were dried, dehydrated and e m b e d d e d in Epon. Thin sections were stained with uranyl acetate and lead nitrate. For light microscopy, 0.5--1 pm thick sections were stained with toluidine blue (1% in 2.5% sodium carbonate, pH 11.1 for 30--120 min). RESULTS
Equilibrium density centrifugation In order to establish a basis for the centrifugation technique, the b u o y a n t density of mast cells was studied in relation to other cells in the peritoneal suspensions by gradient centrifugation on Percoll under equilibrium conditions. Cell suspensions from one rat containing a b o u t 106 mast cells and 20 X 106 total cells suspended in HA were layered on Percoll gradients prepared by high speed centrifugation. A Sorvall HB4 swing-out rotor was used. Centrifugation was performed at 500 × g for 30 min. Equilibrium conditions were checked conveniently with beaded density markers. Good separation of the mast cells from other nucleated cells was easily obtained. Red blood cells, on the other hand, often sedimented with the mast cells. A constant finding was that mast cells from y o u n g animals had consistently lower buoyo
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Fig. 1. Equilibrium density gradient centrifugation on a continuous Percoll gradient generated by high speed centrifugation. Cell suspension from a young rat, weighing 250 g. Gradient ($), mast cells (D), other nucleated cells (x), red blood cells (o). Fig. 2. Equilibrium density gradient centrifugation as in Fig. 1. Cell suspension from an old rat, weighing 500 g. Symbols as in Fig. 1.
139
ant density than mast cells from old rats. The results of t w o typical experiments are shown in Figs. 1 (young rat) and 2 (old rat). The mast cells had a density between 1.09 and 1.15 in the y o u n g rat and between 1.11 and 1.17 in the old rat. In both instances the red blood cells showed a narrow density range of 1.10--1.12 with a peak at 1.11. The results in Figs. t and 2 demonstrate clearly that mast cells m a y be readily separated from nucleated cells b y centrifugation based on density alone. Especially in y o u n g rats, however, good separation from red blood cells cannot be expected by a centrifugation technique based u p o n density only.
Purification o f peritoneal mast cells: step I Based on the above, the following simple technique was devised for purification of mast cells from peritoneal cell suspensions uncontaminated with blood. An aliquot of 0.75 ml of the cell suspensions in HA was added to 3.5 ml Percoll isotonic solution resulting in a final density of 1.110. The solution was mixed thoroughly and allowed to settle. Then, 0.5 ml 0.1% HA was layered on t o p of the solution and the sample was centrifuged at 125 X g for 15 min. The t w o uppermost ml were removed together with 0.5 ml HA used for washing the wall of the test tube. The remaining volume containing the mast cells was washed twice in 0.1% HA (centrifugation at 300 X g and for 5 min). The results of the procedure are shown in Table 1. Four animals in each of three age groups were used because of the marked influence of age on the density of the mast cells. The Table shows satisfactory purity and recovery. The suspensions contained few red blood cells. The recovery of mast cells was dependent on the age of the animals b u t was also satisfactory for the youngest age group (72%).
TABLE 1 Mast cell p u r i f i c a t i o n b y d e n s i t y g r a d i e n t c e n t r i f u g a t i o n (step I). R e s u l t s are m e a n s + S.E.M. Age (days) a
45 62 102
Body weight (g)
200-+3 311-+4 3 9 2 -+ 4
Cell c o n c e n t r a t i o n (%) MC b
R B c.
OC d
Mast cell yield (%)
91+1 92-+1 9 3 -+ 1
7+1 4-+1 4 -+ 1
3-+0 5-+1 5 -+ 0
72-+2 85-+4 100 -+ 6
a T h r e e e x p e r i m e n t s o n 4 d i f f e r e n t rats in e a c h group. b Mast cells, c r e d b l o o d cells, d o t h e r cells.
140
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Fig. 3. Velocity gradient centrifugat!on (40 x g, 5 min) with added red blood cells. Shaded columns are mast cells and open cqlumns red blood cells.
Purification of peritoneal mast cells: step H The previously described results (Table 1) showed that isolation of mast cells with high yield and purity could be achieved by a simple centrifugation technique based on density, provided that the cell suspensions were not contaminated with blood. The 12 consecutive experiments illustrated in Table 1 showed that contamination with blood cells could be avoided to a large extent. However, for cases when such contamination occurred, we devised a method for removing red blood cells. Since a separation based on density
TABLE 2
Mast cell purification by combined density (step I) and velocity gradient centrifugation (step II). Cell suspensions from 6 rats aged 57 -+ 1 days. Red blood cells added to cell suspensions. Results are means + S.E.M. Cell c o n c e n t r a t i o n (%)
Step I
Step II
Mast cells Red blood cells Other cells
65 -+ 2 33 -+ 2 2 -+ 0
98+1
Mast cell yield Total mast cell yield
78 + 2
1_+0 1_+0 82-+7 63+4
141
was n o t feasible, a velocity gradient type of centrifugation procedure was selected. A continuous linear Percoll gradient with a density of 1.020-1.030 was prepared with a peristaltic pump. Mast cell suspensions containing red blood cells were layered on t o p of the gradient and centrifuged for 5 min at 40 X g (HB 4 swing-out rotor). The result of an experiment employing analysis in 3 ml fractions is shown in Fig. 3. It is evident that mast cells could be separated from the red blood cells with this t y p e of centrifugation procedure. As a standard technique we used a gradient with a volume of 36 ml with a density ranging from 1.02 to 1.03 on a cushion of 2 ml Percoll, density 1.12. Two ml of a cell suspension was layered on t o p of this gradient. After centrifugation (40 X g for 5 min), 25 ml of the suspension (containing the red blood cells) was removed by pumping from the top of the tubes. The remaining solution was washed twice in 0.1% HA and resuspended in a desired volume of HA. Results of the complete separation procedure performed on 6 young rats to the peritoneal cell suspensions of which red blood cells had been added is shown in Table 2. These results show clearly that blood cells were removed b y the second step of the procedure. The total mast cell yield was satisfactory. Heparin and 5-HT content o f mast cells
In order to check possible selection of mast cells during the centrifugation procedure, or leakage of amines, cytofluorometric quantitations of 5-HT and heparin were performed on peritoneal mast cells before and after purification. For this experiment the complete purification procedure was used. We analysed mast cell suspensions from 3 rats. The results in Table 3 demonstrate that both the 5-HT and heparin content were unaffected by the purification procedure. Mast cell secretion studies
Mast cell secretion was studied in purified mast cell suspensions from Sprague--Dawley rats using p o l y m y x i n B as a secretagogue. The results shown in Fig. 4 demonstrate that the cells reacted p r o m p t l y with amine secretion. The reactivity, in terms of histamine release was of similar magnitude to that which we previously found in mast cells of crude peritoneal cell suspensions (Gustafsson and Enerb~ick, 1980). IgE~lependent mast cell secretion was studied in h o o d e d Lister rats immunised with egg albumin according to a technique optimised for production of IgE antibodies (Karlsson, 1978). The results of two experiments involving secretion stimulation by challenge with the antigen are shown in Fig. 5. The resulting maximum histamine release was 30--50% which indicates that the reactivity of the mast cell towards immunological challenge was n o t reduced b y the purification procedure. Spontaneous histamine release was low in all three experiments (3--7%).
142 TABLE 3 5 - H y d r o x y t r y p t a m i n e and h e p a r i n c o n t e n t o f m a s t cell p o p u l a t i o n s b e f o r e and a f t e r purif i c a t i o n ( c o m b i n e d p r o c e d u r e , step I + II). Cell s u p e n s i o n s a
5-Hydroxytryptamine b
Heparin b
1 Crude Pure 2 Crude Pure 3 Crude Pure
105 108 131 137 106 116
171 175 164 164 158 163
-+ 4 -+ 3 -+ 5 + 4 -+ 3 + 3
-+ 3 -+ 3 -+ 3 -+ 3 + 4 + 4
a Crude p e r i t o n e a l cell s u s p e n s i o n s c o n t a i n i n g 3--5% mast cells and purified m a s t cell s u s p e n s i o n s c o n t a i n i n g 9 1 - - 9 6 % m a s t cells f r o m each o f 3 d i f f e r e n t rats. b Measured b y m i c r o s c o p e f l u o r o m e t r y in p o p u l a t i o n samples o f 200 m a s t cells. Populat i o n m e a n -+ S.E.M,
Viability test using fluorescein diacetate (FDA ) Crude or purified mast cell suspensions were incubated in solutions of 2 pM FDA in HA. Fluorescence developed rapidly in the mast cells and after 10 min incubation at room temperature, more than 98% of the mast cells in crude and purified mast cell suspensions showed a strong fluorescence.
Morphology o f isolated mast cells Morphology as determined by both light microscopy and electron microscopy was extremely well preserved in mast cells purified by the centrifuga/ P.................
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Fig. 4. A m i n e release f r o m purified m a s t cells i n c u b a t e d w i t h p o l y m y x i n B at 36°C. A m i n e release was assayed in t e r m s o f release o f radiolabelled 5-HT in m a s t cell suspensions f r o m rats i n j e c t e d 24 h prior t o sacrifice w i t h 100 pCi 3H-labelled 5-HT s u b c u t a n e ously.
Fig. 5. IgE-dependent mast cell secretion by challenge with egg albumin in rats immunised for IgE production 2 weeks before sacrifice.T w o separate experiments.
143 iii
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Fig. 6. Electron micrographs of mast cells isolated by gradient centrifugation on Percoll. Endocytotic vesicles containing Percoll particles indicated by arrows. ] ]= 1 pm (upper two figures) and 0.36 pm (lower two figures).
tion procedure. However, by electron microscopy purified mast cells were occasionally found to contain small electron-dense particles enclosed within endocytotic vesicles in the peripheral parts of the cells (Fig. 6). These particles were similar in size to Percoll particles, 10--20 nm (Pertoft et al., 1978). DISCUSSION
The results reported in this paper show that mast cells can be isolated with high yield and purity by centrifugation on gradients of Percoll. This medium, consisting of PVP-coated silica particles has properties which makes it highly
144
desirable for cell separation purposes (Pertoft et al., 1978). Of special importance is its extremely low osmotic pressure, which makes it possible to prepare gradients with high density in various physiological media, and its property of forming continuous gradients during high speed centrifugation. Such continuous Percoll gradients were used with isopycnic centrifugation under equilibrium conditions to determine the b u o y a n t density of the mast cells. We found that mast cells had a higher density than other nucleated cells in crude peritoneal cell suspensions. Moreover, mast cells from young rats had consistently lower density than mast cells from older animals. We found density values for mast cells ranging from 1.09 to 1.17 g/ml. The b u o y a n t density of mast cells has previously been studied by Pretlow and Cassady (1970) w h o reported density ranging from 1.08 to 1.29 in another strain of rats. We have previously demonstrated that dry mass, protein, heparin and amine content all increase in peritoneal mast cells as a function of age in growing rats (Enerb~ick and Mellblom, 1978; Mellblom and Enerb~ick, 1979). Since mast cells are elements with long lives, such differences between y o u n g and old rats are at least to some extent related to aging of the mast cells (Mellblom, 1979). Mast cells thus had a higher b u o y a n t density than other nucleated cells in the crude peritoneal cell suspensions. The red blood cells showed densities between 1.10 and 1.12, overlapping the low density ranges of mast cells. In y o u n g rats there was also some overlap between mast cells and other nucleated cells in the peritoneal cavity. In spite of this, peritoneal mast cells could be isolated with high yield and purity from the crude peritoneal cell suspensions with a simple centrifugation procedure based on density differences. The procedure also gave satisfactory results in cell suspensions from young animals. For the infrequent occasions when peritoneal cell suspensions are contaminated with blood, red blood cells can be eliminated by a subsequent brief centrifugation of velocity gradient type. The two cell types separate owing to differences in cell size rather than differences in density. The morphology of mast cells was excellently preserved after centrifugation on Percoll, judged both by light microscopy and ultrastructural criteria. However, limited uptake of Percoll particles appeared to take place by an endocytotic mechanism. Since this is most probably a temperature dependent p h e n o m e n o n it could possibly have been avoided by lowering the temperature. However, mast cells isolated on Percoll were well preserved with respect to chemical composition, viability and secretory capacity, both after stimulation with polymyxin and with antigen. The cytofluorometric measurements on individual mast cells of crude and purified suspensions further demonstrated that amine leakage, which appears to occur frequently with other isolation procedures, did not occur with the present method. Heparin, which is a matrix c o m p o n e n t of mast cell granules, was likewise not affected b y the purification procedure, indicating that a loss of mast cell granules did n o t occur, and also, something which is most important, that selection of mast cells with respect to heparin content or mass (see Mellblom, 1980) did
145
n o t occur during the centrifugation procedure. Another indication of good preservation of the mast cells was the finding of very low spontaneous amine release in the secretion studies of isolated mast cells. The simple one step centrifugation procedure for isolation of mast cells appears to be ideally suited for preparation of cells for secretion studies in vitro. ACKNOWLEDGEMENT
This work was supported b y grants from the Swedish Medical Research Council, Project No. 2235. REFERENCES And~n, N.E. and T. Magnusson, 1967, Acta Physiol. Scand. 69, 87. Bach, M.K., K.J. Block and K.F. Austen, 1971, J. Exp. Med. 133,752. Cooper, P.H. and D.R. Stanworth, 1976, in: Methods in Cell Biology, Vol. 14, ed. P.M. Prescott (Academic Press, New York) p. 365. Enerb~ick, L., 1974, Histochemistry 4 2 , 3 0 1 . Enerb~/ck, L. and L. Mellblom, 1978, Cell Tiss. Res. 187,367. Enerb~/ck, L. and U. Wingren, 1980a, Histochemistry 6 6 , 1 1 3 . Enerb~/ck, L. and U. Wingren, 1980b, J. Histochem. Cytochem., in press. Enerb~/ck, L., B. Gustafsson and L. Mellblom, 1977, J. Histochem. Cytochem. 27, 32. Glick, D., S.J. Bonting and D. BenBoer, 1956, Proc. Soc. Exp. Biol. Med. 92,357. Gustafsson, B. and L. Enerb~/ck, 1980, Exp. Cell Biol. 48, 15. Jacobsen, C.F. and K. LindenstrSm-Lang, 1940, Acta Physiol. Scand. 1,149. Johnson, A.R. and N.C. Moran, 1966, Proc. Soc. Exp. Biol. Med. 1 2 3 , 8 8 6 . Karlsson, T., 1978, in: Immunoglobulin E and the Mast Cell, Doctoral Thesis (Uppsala University, Uppsala) p. 26. Keller, R., 1966, in: Tissue Mast Cells in Immune Reactions (American Elsevier, New York) p. 50. Lagunoff, D., 1975, in: Techniques of Biochemical and Biophysical Morphology, Vol. 2, eds. D. Glick and R.M. Rosenbaum (Wiley-Interscience, New York) p. 2 8 3 . . Mellblom, L., 1979, Growth Related Changes of Connective Tissue Mast Cells, LinkSping University medical dissertation No. 82. Mellblom, L., 1980, Cell Tiss. Res., in press. Mellblom, L. and L. Enerb~/ck, 1979, Histochemistry 6 3 , 1 2 9 . Padawer, J. and A.S. Gordon, 1955, Proc. Soc. Exp. Biol. Med. 88, 29. Pertoft, H. and T.C. Laurent, 1977, in: Methods of Cell Separation, Vol. 1, ed. N. Catsimpoulas (Plenum, New York) p. 25. Pertoft, H., K. Rubin, L. Kjell~n, T.C. Laurent and B. Klingeborn, 1977, Exp. Cell Res. 110,449. Pertoft, H., T.C. Laurent, T. L~i~isand L. K~igedal, 1978, Anal. Biochem. 88, 271. Pretlow, T.G. and J.M. Cassady, 1970, Am. J. Pathol. 6 1 , 3 2 3 . Sullivan, T.J., K.L. Parker, W. Stenson and C.W. Parker, 1975, J. Immunol. 114, 1473. U v n ~ , B. and I.-L. Thon, 1961, Exp. Cell Res. 23, 45.