Printed in Sweden Copyright 0 1978 by Academic Ress, Inc. All rights of reproduction in any form reserved C014-4827/78/[email protected][email protected]
Experimental Cell Research I1 I (1978) 245-251
T. A. W. SPLINTER,’
and A. VAN BEEK
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, and Laboratory of Experimental and Clinical Immunology, University of Amsterdam, Amsterdam, The Netherlands
SUMMARY Incubation in culture medium at 37°C of normal mononuclear leukocytes, isolated on Ficoll-Isopaque, resulted in a general decrease in the specific gravity of these cells. Monocytes and lymphocytes could then be recognized as separate populations in the density distribution profile. This decrease of the celI density was inhibited by lowering the temperature to 0°C or by the addition of Ficoll-Isopaque to the incubation medium. After pre-incubation at 37”C, renewed contact between mononuclear leukocytes and Ficoll-Isopaque induced a general increase of the cell density. Monocytes and thymidine-incorporating lymphocytes were affected to a greater extent than small lymphocytes. The increase of the specific gravity of monocytes was time and temperature dependent. The density shift of the thymidine-incorporating lymphocytes was only time dependent. The change in the density of small lymphocytes was dependent on the combination of contact with Ficoll-Isopaque and centrifugal stress. Isopaque was the agent responsible for the effects. It is concluded that the use of small molecular substances such as Isopaque as a constituent of density gradients may introduce reversible changes of the specific gravity of mononuclear leukocytes which may limit the separation of mononuclear sub-populations.
Isopyknic centrifugation on Ficoll-Isopaque gradients is widely used to separate cell populations with different densities and volumes [ 1,2]. This treatment may lead to a slight increase of the density of small lymphocytes  and this change may be reverted by a subsequent incubation in culture medium . The purpose of the present report was two-fold. (1) To establish the conditions by which incubation of mononuclear blood cells with a Ficoll-Isopaque mixture leads to a change in the density distribution of i Present address: University Hospital of the University of Amsterdam, Wilhelmina Gasthuis, Department of Internal Medicine, le Helmersstraat 104, Amsterdam, The Netherlands.
these cells and by which this effect may be reverted. (2) To determine whether or not this change in cellular density is restricted to a particular sub-population of the mononuclear leukocytes. MATERIAL
Chemicals Ficoll (uolvsucrose. mol. wt 400000) was obtained from Pharmacia, Uppsala, and Isopaque (sodium, calcium and magnesium N-methylJ,5-diacetamide-2,4,6triiodobenzoate) from Nyegaard & Co., Oslo. RPMI1640 and Heoes were purchased from Flow Laboratories. Avrshire. Scotland: fetal calf serum (FCS) from Gibco, Grand Island, N.Y.; and glutamine‘ from Merck. Darmstadt. GFR. TsHlMethvl thymidine (spec. act. 17’Ci/mmole) was ob&ined from the Radiochemical Centre, Amersham, and phytohaemagglutinin yot;onrt. no. NR-10) from Burroughs Wellcome, ------I
Res 111 (1978)
Splinter et al.
Isolation of mononuclear blood cells Mononuclear cells were isolated from defibrinated blood, as described previously . In brief, detibrinated blood of healthy donors was mixed with an equal volume of tissue culture medium consisting of RPMI-1640 supplemented with 10% (v/v) fetal calf serum (FCS), glutamine (0.03 % (w/v), penicillin (100 W/ml), streptomycin (100 pg/ml) and Hepes (12.5 mM). Of this suspension 25-30 ml were layered on 12 ml of a Ficoll-Isopaque mixture (d= 1.079 g/cm3 at 22°C). After centrifugation (loo0 g, 20 mitt, 22”C), the lymphocytes were collected from the interface and washed once with an equal volume of tissue culture medium. The cells were then resuspended in the culture medium and counted electronically (Coulter Counter, model ZF). The yield was 6O-g0% of the mononuclear cells present in the detibrinated blood. Contamination with granulocytes was less than 5%, and the number of erythrocytes was smaller than or equal to the number of mononuclear cells. For each experiment, blood of a different donor was used.
Gradients and density profiles Continuous gradients were prepared from two stock solutions of Ficoll-Isopaque with densities of 1.050 and 1.085g/cm3, respectively, in siliconized glass tubes (0 1.4 cm, height 12 cm), as described previously . Discontinuous gradients were prepared by careful layering of solutions of Ficoll-Isopaque with densities of 1.050, 1.062 and 1.092 g/cm3, in glass Kimax tubes (0 2.86 cm, height 12.2 cm). Each layer consisted of 4 ml. The density distribution profiles of mononuclear leukocytes were virtually identical for cell loads ranging from 0.5 to 5X 10s cells/gradient. Overloading occurred at cell loads greater than 5 x 106cells/gradient. Therefore, not more than 3.5~ lo5 cells were layered on the continuous gradients. Isopyknic equilibrium of the small lymphocytes was obtained after 15 min at 2200 g and 4”C, as described previously [2, 71. The contents of the tube, containing the linear gradient, were collected by suction through a catheter from the bottom and distributed over 24 fractions of 0.5 ml each. A sample of 0.2 ml was removed for electronic cell counting (Coulter Counter, model ZF). When different numbers of cells were layered upon the gradients, the sample size was so adjusted (0.1, 0.2 or 0.4 ml) that cell countings were comparable. At regular intervals the refractive index of 4-5 fractions was measured and the linearity of the correlation between refractive index and fraction number was checked with the method of least squares. The density of each fraction was computed from the relationship between the density at 4°C and the refractive index at 25°C . The separated cells in the discontinuous gradient were harvested by careful collection of the different layers with a Pasteur pipette. The average yield of cells from both gradients was 85% (S.D. 6%, n=19).
Size distribution and percentage of monocytes The percentage of monocytes was determined from the size distribution of the mononuclear cells according to Exp Cd Res 1I I (1978)
Loos et al. . In brief, the following method was used. Contaminating erythrocytes in the mononuclear cell suspension were lysed by isotonic NH&l solution . Next, 0.2 ml of this suspension, containing 1X 106 cells, was diluted with 20 ml of particle-free phosphatebuffered saline. This sample was introduced into a Coulter Counter (model ZF), connected to a pulseheight analyser (Channelyzer, model C-100). Optimal conditions were as follows: the manometer had a 100 pm orifice; the attenuator was set at 2, the aperture at 16, the threshold at 10, and the window width at 100. Size distribution curves were obtained by plotting the counts of each channel as a percentage of the total count against the channel number. To determine the percentage of monocytes the lower threshold was set at the lowest point between the two peaks in the size distribution profile. The total count between this channel and channel 99 was integrated and expressed as a percentage of the total counts between channels 4 and 99.
[3H]Thymidine incorporation For the analysis of the density distribution of DNAsynthesizing cells, suspensions were pre-incubated at 37°C for 3 h with [3H]TdR (2 &i/ml, spec. act. 17 Ci/ mmol) in tissue culture medium (consisting of RPMI1640 supplemented with 10% (v/v) FCS, glutamine
Fig. 1. Abscissa: density (g/cm3); ordinate: no. of cells/fraction (% of total recovered). X-X, 0; O-O, 90; O--0,180; A-A, 240 min. Effect of incubation at 37°C in culture medium on the density distribution of mononuclear cells.
Table 1. Identification
of a separate population
Unseparated cells % of monocytes Cells with a density > 1.050g/ml < 1.062 g/ml % monocytes within the light cell fraction Cells with a density > 1.062 g/ml < 1.092 g/ml % monocytes within the heavy cell fraction Recovery after separation
cell density changes
r=O min (%)
t=90 min m
t= 180 min (%I
(% of cells recovered)
(% of cells recovered)
(% of cells recovered)
Mononuclear cells were incubated at 37°C in culture medium. At various times the cells were separated into two populations on a discontinuous gradient of Ficoll-Isopaque. The percentage of monocytes was determined in the unseparated population and in both separated populations.
(0.03 % w/v), penicillin (100 III/ml), streptomycin (100 &ml) and Hepes buffer (12.5 mM, pH 7.4)). The [$H]TdR uptake was stopped by adding thymidine (2 n&ml) and storing the samples at 0°C. The cells were collected on glass fibre filters (Gelman Instr., type A), washed carefully with demineralized water, dried and counted for 10 min in 2 ml of a scintillation mixture (15 g PPO, 1 g POPOP, 2.5 1 Toluene) in a liquid scintillation counter according to Schellekens & Eijsvoogel.
Lymphocyte stimulation by PHA was performed as described by Schellekens & Eijsvoogel.
RESULTS Effect of incubation on the density distribution offreshly isolated mononuclear leukocytes
After isolation of mononuclear leukocytes from blood with the aid of a Ficoll-Isopaque gradient according to Bsyum ([ 11,see Methods), the cells were incubated in culture medium at 37°C in a concentration of 5X lo6 cells/ml. The density distribution of these cells was determined after various time intervals (fig. 1). In addition, a part of the same cell suspension was separated after 0, 90 and 180 min on a discontinuous gradient
consisting of Ficoll-Isopaque layers with densities of 1.050, 1.062 and 1.092 g/cm3. The separated cells were harvested, counted and differentiated; the results are summarized in table 1. There was a gradual decrease of the mean cell density during 4 h of incubation from 1.071 to 1.067 g/cm3 (see fig. 1). Moreover, a separate population of cells with a density of less than approx. 1.064 g/cm3 appeared. As shown in table 1, this separate population (1.050 glcm3
Splinter et al.
Fig. 2. Abscissa: density (glcm3); ordinate: no. of cells and incorporated [3H]TdR/fraction (both as % of total recovered). Ficoll-Isopaque-treated cells; X-X, unlabeled; O---O, labeled; control cells: A-A, unlabeled; q - - -0, labeled. Effect of incubation in culture medium, containing Ficoll-Isopaque, for 3 h at 37°C on the density distribution of [SH]TdR labeled and unlabeled mononuclear cells.
[3H]thymidine ([3H]TdR) to label the DNAsynthesizing lymphocytes, which are morphologically defined as ‘atypical lymphocytes’ . Again, the mean density of the total population had shifted from 1.070 to 1.068 g/cm3 after 3 h at 37°C. The,mean density of the labeled lymphocytes decreased from 1.067 g/cm3 after 90 min to 1.063 g/cm3 after 180 min. No separate population of light cells could be recognized in the density profiles (not shown). It was concluded that incubation at 37°C in culture medium caused a general decrease of the density of all types of freshly isolated mononuclear leukocytes resulting in the appearance of a separate population of monocytes. Exp Cell Res 11 I (1978)
Fig. 3. Abscissa: density (g/cm3); ordinate: no. of cells/fraction (% of total recovered). x-x, Control cells: C-0, 37°C;
cells; Ficoll-Isopaque-treated O-O,
Effect of incubation in Ficoll-Isopaque for 30 min at different temperatures on the density distribution of mononuclear cells.
Factors influencing the shijt of the density distribution profile It was tested whether the described density shift could be affected by the presence of Ficoll-Isopaque or the temperature during the incubation. The effect of Ficoll-Isopaque was studied by comparing the density distribution profiles of mononuclear leukocytes incubated either in a 1: 1 mixture of culture medium and PBS or in a 1: 1 mixture of culture medium and a Fitoll-Isopaque solution (d = 1.077 g/cm3). The mixtures were incubated at 37°C for 3 h in the presence of [3H]TdR. The density of the labeled and unlabeled lymphocytes incubated in the presence of Ficoll-Isopaque remained higher than the density of the
Isopaque-induced cell density changes
density (g/cm3); ordinate: no. of cells and incorporated [sH]TdR/fraction (both as % of total recovered). Control cells: X-X, unlabeled; Cl-- -0, labeled. Ficoll-Isopaque-treated cells: A-A, unlabeled; 0- - -0, labeled. Effect of incubation in Ficoll-Isopaque for 30 min at WC on the density distribution of lymphoblasts stimulated by phytohaemagglutinin (PHA).
Fig. 4. Abscissa:
density (g/cm3); ordinate: no. of cells and incorporated [3H]TdR/fraction (both as % of total recovered). Ficoll-Isopaque-treated cells: X-X, unlabeled; X---X, labeled; control cells: O-O, unlabeled; 0- - -0, labeled. Effect of incubation in Ficoll-Isopaque for 30 min at 0°C on the density distribution of [3H]TdR labeled and unlabeled mononuclear cells.
Fig. 5. Abscissa:
same cells in the control mixture. No separate monocyte population could be distinguished in the density profile of the lymphocytes that had been in contact with Ficoll-Isopaque during the incubation at 37°C (fig. 2). The effect of temperature on the reversion of the density shift was tested by comparing the density distribution profiles of mononuclear leukocytes incubated in culture medium at either 0 or 37°C. Again, neither a separate monocyte population nor a density shift of the labeled lymphocytes was found at 0°C (not shown here). From these experiments it was concluded that the shift of the density distribution protile could be interpreted as a reversion of an
increase of the cell density, induced during the isolation procedure of the mononuclear leukocytes. Re-induction of an increase in the density of mononuclear cells To investigate the possibility of re-induction of density changes by Ficoll-Isopaque the following protocol was used. After isolation of the mononuclear cells the effects of the isolation on Ficoll-Isopaque gradients were reversed by a pre-incubation in culture medium at 37°C for 3 h; [3H]TdR was added to label the blastoid lymphocytes at the same time. (1) In the first experiment four equal cell pellets were prepared; one pellet was reExp Cell Res I II (1978)
Splinter et al.
suspended in culture medium (control) and the other three in 1 ml of an isotonic FicollIsopaque mixture (d=1.040 g/cm”). The density distribution of the control suspension was analysed immediately. The other three suspensions were incubated for 30 min at 0, 25 and 37°C respectively, and then submitted to density analysis. Fig. 3 shows that the extent of separation between monocytes and lymphocytes decreased with the increase of the temperature of incubation. The mean density of the small lymphocytes did not change, whereas the complete density profile of the labeled lymphocytes shifted to a slightly higher value and their mean density changed from 1.064 to 1.067 g/cm3 independently of the temperature (not shown). In two similar experiments it was confirmed that even an incubation of 30 min at 0°C sufficed to produce the above-mentioned effects (fig. 4) and that these effects were recognizable after 10 min of incubation (not shown). (2) It was investigated whether a combination of centrifugal force and contact with Ficoll-Isopaque was required for a change in the density of the small lymphocytes. Small lymphocytes, depleted of monocytes by treatment of the blood with carbonyl-iron , were used. One part of the cells suspended in culture medium was centrifuged as such; the other part was layered on Ficoll-Isopaque (d= 1.079 g/cm3 at 22°C) and then centrifuged (20 min, 1000 g, 4°C). The pellet and the lymphocyte band at the interface of the gradient were harvested separately and washed once with culture medium. Both suspensions were then submitted to density analysis. The total density profile of the cells that had been in contact with Ficoll-Isopaque had slightly shifted towards a higher density (not shown). The mean densities of the control lymphocytes and the cells that had been Exp Cell Res 111 (1978)
in contact with the Ficoll-Isopaque were 1.069 and 1.071 g/cm3, respectively. (3) The separate effect on lymphoblasts was tested with a population of actively proliferating lymphocytes. Lymphocytes were cultured for 3 days in the presence of PHA and labeled with [3H]TdR during the last 3 h in culture. One part of this cell suspension, containing 80 % of lymphoblasts, was resuspended in culture medium and another part in Ficoll-Isopaque (d=1.040 g/ cm3). After incubation at 0°C for 30 min, both cell suspensions were submitted to density analysis (fig. 5). Contact with Fitoll-Isopaque induced an increase in the mean density of the blastoid cells from 1.065 to 1.067 g/cm3. Separate effects of Ficoll, Isopaque and Ficoll-Isopaque
The effect of the separate components of Ficoll-Isopaque was investigated by incubation of mononuclear cells for 30 min at 0°C either in culture medium or in an isotonic Ficoll solution, or in an isotonic Isopaque solution, and in a 1: 1 mixture of both . As in the previous experiments, the mononuclear leukocytes had been pre-incubated for 3 h at 37°C in culture medium in the presence of [3H]TdR. Incubation in Ficoll only, produced no significant shift of the density distribution. Ficoll-Isopaque and Isopaque alone, induced a decrease of the resolution between the separate monocyte and lymphocyte populations and a shift of the mean density of the labeled lymphoblasts from 1.065 to 1.068 g/cm3 (not shown). DISCUSSION The first experiments showed that the specific gravity of mononuclear leukocytes, freshly isolated with the aid of Ficoll-Isopaque according to Boyum [ 11,could be de-
creased by incubation in culture medium at 37°C. This decrease in density was found consistently when different cell preparations were used. Monocytes were affected to a larger extent than iymphoblasts and small lymphocytes; this difference resulted in a separation of monocytes and small lymphocytes. The decrease in density was temperature dependent and could be inhibited by the presence of Ficoll-Isopaque in the incubation mixture. The latter observation suggested that the changes might be considered as a restoration of cell density after contact with Ficoll-Isopaque. This conclusion was supported by the findings that a subsequent contact with Ficoll-Isopaque re-induced an increase of the density of mononuclear leukocytes. Again, the magnitude of these density changes as well as the experimental conditions varied for the different kinds of mononuclear leukocytes. Isopaque was identified as the active component in this respect. Some insight into the mechanism by which Isopaque induced an increase of the specific gravity of cells was derived from the conditions leading to this phenomenon. Since the order of magnitude of the density changes decreased from monocytes via lymphoblasts to small lymphocytes, cellular volume and surface area seemed to influence this phenomenon. In addition, only the increase of the density of monocytes induced by Isopaque was temperature dependent, indicating that, for this cell type,
cell density changes
metabolic processes such as pinocytosis may be actively involved. Therefore, the most likely explanation is that small ionic compounds such as metrizoate (Isopaque) induced a density change in cells either by simple diffusion or by extraction of water as a result of a change in the Donnan equilibrium. With respect to phagocytic cells, active uptake may enhance the magnitude of the density changes. The reversion of the Isopaque-induced density changes is probably energy dependent since it was found to be temperature dependent for all mononuclear leukocytes. These observations stress the absolute need for constant experimental conditions with respect to cell load, centrifugal force and time, as well as temperature, for reproducible analysis of the cellular density with Ficoll-Isopaque gradients. We are grateful to Dr J. A. Loos, Dr D. Roos and Professor Dr F. J. Cleton for their criticism.
REFERENCES 1. Beyum, A, Stand j clin lab invest 21, suppl. 97 (1968) 77. 2. Loos, J A & Roos, D, Exp cell res 86 (1974) 333. 3. Sqp:inter. T A W & Reiss, M, Exp cell res 89 (1974) 4. Loos, H, Blok-&hut, B, Kipp, B, van Doom, R & Meerhof. L. Blood 48 (1976) 743. 5. Schellekkns~ P T A & Eijsvoogel, V P, Clin exp immuno13 (1%8) 571. 6. Wood, T A & Frenkel, E P, Am j med 42 (1%7) 923. 7. Loos, H, Blok-Schut, B, van Doom, R, Hoksbergen, R, Brute1 de la Riviere, A & Meerhof, L, Blood 48 (1976) 731. Received December 14, 1976 Revised version received September 9, 1977 Accepted September 15, 1977
Exp Cell Res Ill