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Partial separation of Sendai virus-fused lymphocytes by velocity sedimentation
Journal of Immunological Methods 5 (1974) 121-129. © North-Holland Publishing Company
PARTIAL SEPARATION OF SENDAI VIRUS-FUSED LYMPHOCYTES BY VELOCITY SEDIMENTATION D.C. ALLISON*, M. RALPH and E.P. COHEN Department of Surgery* and La Rabida-University of Chicago Institute, University of Chicago, Chicago, IlL 60649, U.S.A.
Accepted 7 March 1974
Received 6 February 1974
The partial separation of Sendai virus-fused mouse lymphocytes by velocity sedimentation, a technique that separates cells on the basis of size, is described. In addition, density separation of cultures containing fused cells with iso-osmotic BSA allows study of viable ceils after 72 hr in culture.
1. INTRODUCTION Fusion rates o f primary cultured lymphocytes, even after Sendai virus treatment, are low (reviewed by Poste, 1970). Fused cells also show an exceptionally high mortality in culture (Watkins and Grace, 1967). These problems, combined with the poor culture survival of lymphocytes (Mosier and Pierce, 1972), severely limit the study of fused, non-transformed lymphocytes. We have applied the technique of 'velocity sedimentation' (Miller and Philips, 1969) to increase the percentage of Sendai virus-fused lymphocytes. This technique separates cells on the basis of size. The cells are allowed to sediment through a low-density medium. Larger cells are believed to sediment faster because their lower surface area-to-volume ratio minimizes friction. This method should increase the proportion o f fused cells in a mixture of fused and non-fused parental cells because fused cells initially have twicethe volume of their parents. Isopycnic centrifugation in iso-osmotic bovine serum albumin (BSA) was also performed to remove dead cells. This separation is based on the plasma membrane breakdown o f non-viable cells which increases their density. These methods were applied to the study of the incidence of binucleated cells in Sendai virus-treated cultures of radioactively labeled thymocytes and non-labeled spleen cells. Other groups were treated in an identical manner except that Sendai virus was omitted from the culture. In either case, binucleated cells could not be considered fused if they showed identical labeling patterns in each nucleus.
121
122
D.C. ALLISON, M. RALPH and E.P. COHEN
2. MATERIALS AND METHODS
2.1. Preparation of isopycnic, iso-osmotic BSA The method described by Shortman et al. (1972) was used. An unbuffered saline solution containing 0.168 M NaC1 (121 vol); 0.168 M KC1 (4 vol); 0.112 M CaC12 (3 vol); 0.168 M MgSO4 (1 vol), and 0.168 M KH2PO4 (1 vol) was prepared. BSA (Fraction V, Armour Pharm. Co., Chicago, Ill.)was dissolved in a 15-20% aqueous solution (w/w) and dialyzed at 4°C against four changes of ten volumes each of deionized water. This solution was lyophilized and the BSA was placed over anhydrous P2Os for two days to remove residual water. A 35% stock solution was then prepared by dissolving 100 g of dried albumin in 182 ml of the saline solution and 4 ml of distilled water. This solution was diluted to a final density of 1.0931.094 g/cm a with saline.
2.2. Separation of viable and non-viable cells with isopycnic centrifugation To separate non-viable cells from the suspension, (Shortman et al., 1972) mouse lymphocytes were centrifuged at 100g for 10 rain and suspended in 3 ml of the BSA solution. A Pasteur pipet was used to introduce 2 ml of cell-free BSA under this mixture. A second Pasteur pipet was used to layer 4 ml of saline above the BSA solution. The saline-BSA interface was mixed with the end of this pipet. The tube was then centrifuged at 3500 g for 10 min. Viable cells banded at the BSA-saline interface. Dead cells sedimented to the bottom of the tube.
2.3. Isotopic labeling of mouse lymphocytes and their detection by autoradiography Mouse thymocytes of AKR males 10 weeks of age were labeled with a single intraperitoneal injection (5 /aCi/g body weight) of [a H] thymidine (5 Ci/mM, Amersham-Searle, Arlington Heights, Ill.), four days before sacrifice (Matsuyama et al., 1966). This procedure labels over 90% of mouse thymocytes as detected by autoradiography. Slides with labeled thymocytes were prepared (see 2.8. below) and fixed in cold methanol and dried overnight at room temperature. They were then processed for autoradiography according to the procedure of Kopriwa and Leblond (1962). This method includes two 1 min stops in the following solutions: 100% ethanol; 95% ethanol; 80% ethanol, and distilled water. The slides were dried overnight and dipped into NBT-2 emulsion (Eastman Kodak & Co., Rochester, New York). They were dried for 30 min at 30°C. The slides were then stored in sealed plastic boxes at 4°C with an average exposure time of four weeks. They were developed in D-170 Kodak for 6 min and fixed with Kodak Fixer for 5 min. All processing was performed at 17°C in a light-proof room. After development, the slides were rinsed in tap water for 15 min, dried, and lightly stained with Giemsa.
Separation or fused lymphocytes
123
They were then passed through xylene for 1 hr and covered with coverslips mounted with 'Permount' (Fisher Scientific Co., Pittsburgh, Pa.).
2.4. Growth of Sendai virus, fusion procedure and culture conditions Sendai virus, obtained as a gift from J. Schwaber, was grown on the allantoi~ membranes of 9-day old embryonated chicken eggs. The eggs were inoculated with 0.2 ml of a 10-3 dilution of infected allantoic fluid as described by Klebe et al. (1970). After 72 hr incubation at 35°C, the eggs were placed at 4°C for 12 hr to induce clotting. The infected allantoic fluids were then harvested and virus was assayed by its capacity to agglutinate a suspension of 0.37% chicken red blood cells. The allantoic fluid was clarified by centrifugation at 2000 g for 20 min and virus was pelleted by centrifugation at 16,000g for 1 hr, after which the pellet was resuspended in a 0.5% BSA solution in PBS to 1/lOth the starting volume. After a second clarification, the virus suspension was inactivated with/3-propiolactone. An aqueous solution of fl-propiolactone was diluted to a 2% concentration with a buffered saline solution (100 ml of isotonic saline containing 1.68 g of Na2 CO3 and 0.5 ml of 4% Phenol Red). A virus suspension containing 10,000 hemagglutination units (HAU)/ml was brought to a final concentration of 0.05% /3-propiolactone with this solution. The virus suspension in the same solution was incubated at 4°C with shaking for 10 min and then at 37°C for 2 hr, and lastly, at 4°C overnight to completely inactive the ~3-propiolactone. The virus was diluted once again and stored in ampules at -70°C. For cell fusion, virus ( 2 0 0 - 5 0 0 HAU/2 × 106 cells) was incubated at 4°C with lymphocytes (107/ml) for 10 min in Dulbecco's medium without FCS. The cells were then pelleted by centrifugation at 100g for 10 min to increase cell contact. The pellet was warmed to 37°C for 10 min and then gently dispersed by pipetting. The cells were incubated for 2 hr in 5% CO2 at 37°C and then diluted to the final culture conditions: 2 × 106 cells in Dulbecco's complete medium with 10% FCS.
2.5. Velocity sedimentation Fused cells were partially purified by velocity sedimentation according to the method described by Miller and Philips (1969). Two identical chambers (12.5 cm diameter) were fitted with separate inflow and outflow ports and a common inflow channel for the FCS gradient (fig. 1). This arrangement allowed simultaneous study of two experimental groups under similar conditions. A separation started with the introduction of 30 ml of PBS into ea hch3~amber to minimize surface disturbances at the rising meniscus. The cell samples, in a 30 ml volume of PBS (6% FCS), were then introduced under the saline. This was followed by the slow introduction of 800ml of a linear 15-30% gradient of FCS in PBS. The filling rate was never greater than 40 ml/chamber/min and was lower for the first 150 ml (15 ml/min). The cells were allowed to settle from 2 to 4 hr. Fractions were taken off the
124
D.C. ALLISON, M. RALPH and E.P. COHEN GRADIENT INFLOW CHANNEL
STOPCOCK2
OUTFLOW CHANNEL 2
OUTFLOW CHANNEL 1
Fig. 1. Schematic diagram of velocity sedimentation apparatus. Experimental and control cells are introduced into separate sedimentation chambers through separate inflow channels. An anticonvection gradient (15-30% FCS in PBS) is slowly introduced underneath the cells which then are allowed to settle for 2 - 4 hr. Fractions are taken off the bottom of each chamber through the outflow channels. Larger cells, which have a greater sedimentation velocity, are in the earlier fractions. b o t t o m at a rate similar to the rate at which the chambers were filled. (The more rapidly sedimenting cells would be found in the earlier fractions.) The entire separation procedure was carried out at 4°C.
2.6. Cell preparation and media Thymus and spleen cells were pressed through a screen into cold PBS. Debris was allowed to settle for 10 min and the cells in the supernatant then pelleted in another tube. This pellet was resuspended in 'Tris buffered' NH4C1 (Boyle, 1968) for 7 min at 4°C (Shortman et al., 1972). The cells were then pelleted by centrifugation for 8 rain at 100g, washed twice in cold PBS, counted, and placed in the appropriate media. Cell culture was carried out in Dulbecco's Complete Medium (GIBCO, Grand Island, New York) supplemented with: L-glutamine (290mg/1), penicillin (100 units/ml), streptomycin (100/~g/ml), thymidine (10 -4 mM/ml), and 10% FCS. CF outbred mice were kindly provided by The University of Chicago Animal facilities. A K R and A/J mice were obtained from Jackson Laboratory, Bar Harbor, Maine.
2. 7. Preparation of cells for determination of radioactivity by scintillation counting Cells were radioactively labeled during a 2-hr incubation in Dulbecco's medium to which 5 mCi/ml [3 H] thymidine was added. After incubation they were centrifuged and the cell pellet was resuspended in 5% trichloroacetic acid at 4°C. This precipitate was caught on Whatman GF/C fiber filters. The filters were washed
Separation or fused lymphocytes
125
with 10 ml of cold methanol and vacuum-dried for 2 hr at 90°C after which they were placed in Liquifluor and the radioactivity in the filters was detected in a liquid scintillation counter. This method gave a linear correlation between cell number and radioactivity over a 7-fold dilution.
2.8. Preparation of cells for examination Cells were sedimented with the aid of a Cyto-Centrifuge (Shandon Southern Inst., Ltd., Camberly Surrey, England). Approximately 0.5 ml of cells (106/ml) were introduced into a centrifugation block and the cells then centrifuged for 5 min onto glass slides at 150g. The slides were fixed in cold methanol for 7 min, processed for autoradiography, and stained. Cells for the detection of 'streaming' were counted with a Coulter Counter (Coulter Electronics, Hialeah, Florida). All other cells counts were performed using a hemocytometer. Coded specimens were examined for frequency of binucleated and fused cells. A binucleated cell was considered to be a fused cell if it had at least 7 grains over one nucleus and none over the other. Statistical analysis was performed using the Chi Square method of analysis.
2.9. Mixed immune hemagglutination The procedure of Watkins and Grace (1967) was used. 'Sensitized Sheep Red Blood Cells' (SRBC) were prepared by incubating SRBC first with mouse antiSRBC antiserum and then rabbit anti-mouse IGG antiserum (Miles Lab., Kankakee, Ilk). Mixtures of AKR thymocytes and A/J spleen cells reacted with anti A/J antiserum from AKR mice. These cells were then incubated for one hour with the sensitized SRBC'. Slides were prepared in the usual manner.
3. RESULTS
3.1. Separation of viable cells by density gradients More than 90% of viable cells present in mixed cultures of thymocytes and spleen cells 2 4 - 7 2 hr after fusion were recovered in density gradients of BSA. Ninety-nine percent of these recovered cells excluded Trypan Blue. Fused cells were recovered by this method 72 hr after fusion (fig. 2).
3.2. Velocity sedimentation of thymocytes above and below the 'streaming limit' The 'streaming limit' is the cell concentration (3 × 106/ml) above which cells do not sediment independently. This cooperative streaming effect causes the cells to sediment rapidly. It is not known if size dependent sedimentation occurs under streaming conditions. To demonstrate streaming effects 5 × 109 thymocytes were
126
D.C. ALLISON, M. RALPH and E.P. COHEN
ill
P
Fig. 2. A fused l y m p h o c y t e . [ 3 H ] t h y m i d i n e - l a b e l e d A K R t h y m o c y t e s were fused with nonlabeled A/J spleen cells. Fused cells in this population were purified by velocity sedimentation and dead cells eliminated from culture by density centrifugation. A binucleated cell with one labeled nucleus was considered fused if it showed 7 or more grains in the labeled nucleus. 100 !,JJ 9 0 80-
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20-
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18
16
14
12
SEDIMENTATION
10
8
6
VELOCITY
4
2
(mm/hr)
Fig. 3. D e m o n s t r a t i o n o f t h e 'streaming' p h e n o m e n o n . A 3-hr velocity sedimentation was performed with 5 X 109 CF t h y m o c y t e s (e) in one sedimentation chamber and 5 X 107 (A) in t h e other. The more rapid sedimentation o f t h e 5 X 109 cells is apparent.
Separation of fused lymphocytes
127
placed in one sedimentation chamber and 5 × 107 thymocytes in the other. A 3 hr sedimentation was performed under identical conditions for both chambers (fig. 3). The higher cell concentration exceeded the 'streaming limit' of the chamber by at least a factor of 50. The more rapid sedimentation of these cells was apparent.
3.3. Separation or fused lymphocytes above the streaming limit The streaming phenomenon would seem to limit the total number of cells that can be separated by velocity sedimentation. To determine if it was possible to partially purify fused cells at concentrations above which streaming takes place, [3 H] thymidine-labeled AKR thymocytes were mixed with non-labeled A/J spleen cells. One-half of the mixture was exposed to Sendai virus and the remainder of the mixture was treated in a similar manner except that Sendai virus was omitted. Approximately, 2 X 109 cells were placed in each sedimentation chamber and separation performed over a 3~ hr period. As controls, non-separated groups were kept under similar conditions. The most rapidly sedimentating cells (10% of the total)
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SEDIMENTATION VELOCITY ( m m / h r )
Fig. 4. Purification o f binucleated cells beneath the streaming limit. Sendai virus-treated CF t h y m o c y t e s purified above the streaming limit were purified a second time beneath the streaming limit. T h e peak o f binucleated cells (A) at 5.4 m m / h r occurred before t h e radioactively labeled single cell peak (e) and all other velocities studied (p < 0.01).
128
D.C. ALLISON, M. RALPH and E.P. COHEN
Table 1 Partial purification of binucleated and fused cells in a mixture of labeled thymocytes and nonlabeled spleen cells by velocity sedimentation above the streaming limit.
Sendai virus
Group 1
Group 2
Group 3
Group 4
-
+
-
+
Separation by velocity sedimentation
-
+
+
No. cells studied
3600
2322
4200
7400
No. and percentage of binucleated cells*
18 *0.50%)
12 (0.52%)
47 (1.12%)
85 (1.15%)
No. and percentage of fused cells**
0
1 (0.04%)
0
12 (0.16%)
* Labeling pattern of both nuclei identical. ** Binucleated cell with one unlabeled nucleus and the other with 7 or more autoradiographic grains.
were compared with these controls. After elimination of non-viable cells by BSA gradients, the cells were processed for the mixed immune hemagglutination reaction (Watkins and Grace, 1967). Autoradiographs of cells from the following groups were examined for the incidence o f binucleated and fused cells: 1) non-separated, non-virus-treated cells; 2) non-separated, virus-treated cells; 3) separated, non-virus-treated cells; and 4) separated, virus-treated cells. Fused cells as detected by autoradiography were found only in the Sendai virus-treated groups (p < 0.01, fig. 4) (table 1). Binucleated cells occurred in both Sendai- and non-treated groups, with only a slight increase in the Sendai treated-group. Separation o f the cells above the 'streaming limit' led to a 2 - 3 - f o l d increase in the incidence o f binucleated and fused cells (t9 < 0.01) (table 1).
3.4. Separation of binucleated cells beneath the streaming limit 3 H-Labeled CF1 thymocytes were treated with Sendai virus and put into culture for 12 hr. An initial separation was performed above the streaming limit. A study of the most rapidly sedimenting 12% o f the cells revealed a 2 - 3 - f o l d increase in binucleated cells (approximately 1.5/1000 to 3.7/1000). After elimination of nonviable cells by density gradient separation, this rapidly sedimentating fraction was further purified by velocity sedimentation at concentrations beneath the streaming limit (fig. 4). Several thousand cells were studied for the incidence of binucleated cells at selected velocities. A maximum percentage o f binucleated cells occurred at a sedimentation velocity o f 5.4 mm/hr. This peak was significantly higher than the other fractions studied and in the unseparated mixture (p < 0.01). It was also ahead
Separation or fused lymphocytes
129
of the sedimenting single cells measured by scintillation counting. The large amount of radioactivity in rapidly sedimenting fractions was due to the presence of residual cell clumps reflecting selection o f the initial separation.
4. DISCUSSION Velocity sedimentation is an effective way of enriching the proportion of lymphocytes in a mixture of fused and non-fused cells. Density separation with isoosmotic BSA may be used to eliminate non-viable cells and does not select against fused cells. The separation o f cells above the streaming limit may reflect: 1) a size dependent separation; 2) dilution of the cells by streaming through to a concentration beneath the streaming limit with size-dependent sedimentation following, or 3) an association of fused cells with rapidly sedimenting clumps. The last explanation is unlikely as the incidence of binucleated cells is increased in the separated, nonSendal-treated group (p < 0.01). Although much resolution is lost, separation above the streaming limit may be of some value when a low purification of a large number of fused cells is needed. Separation below the streaming limit is the obvious choice when experimental design calls for a high percentage of fused cells. In this work velocity sedimentation significantly lowered the time necessary to detect fused cells. This technique may also aid in the detection o f fused cell products in other systems. The demonstration of a high number of binucleated cells in non-Sendai-treated cultures is unexplained and emphasizes the necessity of having a labeling pattern, or other markers (Watkins and Grace, 1967; Frye a n d Edidin, 1970), to identify fused cells when morphological criteria are not sufficient.
ACKNOWLEDGEMENT This work was supported by a research grant from The Leukemia Research Foundation, Inc.
REFERENCES Boyle, W., 1968, Transplantation 6,761. Frye, L.D. and M. Edidin, 1970, J. Cell Sci. 7,319. Klebe, R.J., T-R. Chen and F.H. Ruddle, 1970, J. Cell Biol. 45, 74. Kopriwa, B.M. and C.P. Leblond, 1962, J. Histochem. and Cytochem. 10, 269. Matsuyama, M.B., M.N. Wiadrowski and D. Metcalf, 1966, J. Exptl. Med. 123,559. Miller, R.G. and R.A. Philips, 1969, J. Cell Physiol., 73, 191. Mosier, D.E. and C.W. Pierce, 1972, J. Exptl. Med. 136. Poste, G., 1970, Advan. Virus Res. 16,303. Shortman, K., N. Williams and P. Adams, 1972, J. Immunol. Methods 1,273. Watkins, J.F. and D.M. Grace, 1967, J. Cell Sci. 2, 193.
PARTIAL SEPARATION OF SENDAI VIRUS-FUSED LYMPHOCYTES BY VELOCITY SEDIMENTATION D.C. ALLISON*, M. RALPH and E.P. COHEN Department of Surgery* and La Rabida-University of Chicago Institute, University of Chicago, Chicago, IlL 60649, U.S.A.
Accepted 7 March 1974
Received 6 February 1974
The partial separation of Sendai virus-fused mouse lymphocytes by velocity sedimentation, a technique that separates cells on the basis of size, is described. In addition, density separation of cultures containing fused cells with iso-osmotic BSA allows study of viable ceils after 72 hr in culture.
1. INTRODUCTION Fusion rates o f primary cultured lymphocytes, even after Sendai virus treatment, are low (reviewed by Poste, 1970). Fused cells also show an exceptionally high mortality in culture (Watkins and Grace, 1967). These problems, combined with the poor culture survival of lymphocytes (Mosier and Pierce, 1972), severely limit the study of fused, non-transformed lymphocytes. We have applied the technique of 'velocity sedimentation' (Miller and Philips, 1969) to increase the percentage of Sendai virus-fused lymphocytes. This technique separates cells on the basis of size. The cells are allowed to sediment through a low-density medium. Larger cells are believed to sediment faster because their lower surface area-to-volume ratio minimizes friction. This method should increase the proportion o f fused cells in a mixture of fused and non-fused parental cells because fused cells initially have twicethe volume of their parents. Isopycnic centrifugation in iso-osmotic bovine serum albumin (BSA) was also performed to remove dead cells. This separation is based on the plasma membrane breakdown o f non-viable cells which increases their density. These methods were applied to the study of the incidence of binucleated cells in Sendai virus-treated cultures of radioactively labeled thymocytes and non-labeled spleen cells. Other groups were treated in an identical manner except that Sendai virus was omitted from the culture. In either case, binucleated cells could not be considered fused if they showed identical labeling patterns in each nucleus.
121
122
D.C. ALLISON, M. RALPH and E.P. COHEN
2. MATERIALS AND METHODS
2.1. Preparation of isopycnic, iso-osmotic BSA The method described by Shortman et al. (1972) was used. An unbuffered saline solution containing 0.168 M NaC1 (121 vol); 0.168 M KC1 (4 vol); 0.112 M CaC12 (3 vol); 0.168 M MgSO4 (1 vol), and 0.168 M KH2PO4 (1 vol) was prepared. BSA (Fraction V, Armour Pharm. Co., Chicago, Ill.)was dissolved in a 15-20% aqueous solution (w/w) and dialyzed at 4°C against four changes of ten volumes each of deionized water. This solution was lyophilized and the BSA was placed over anhydrous P2Os for two days to remove residual water. A 35% stock solution was then prepared by dissolving 100 g of dried albumin in 182 ml of the saline solution and 4 ml of distilled water. This solution was diluted to a final density of 1.0931.094 g/cm a with saline.
2.2. Separation of viable and non-viable cells with isopycnic centrifugation To separate non-viable cells from the suspension, (Shortman et al., 1972) mouse lymphocytes were centrifuged at 100g for 10 rain and suspended in 3 ml of the BSA solution. A Pasteur pipet was used to introduce 2 ml of cell-free BSA under this mixture. A second Pasteur pipet was used to layer 4 ml of saline above the BSA solution. The saline-BSA interface was mixed with the end of this pipet. The tube was then centrifuged at 3500 g for 10 min. Viable cells banded at the BSA-saline interface. Dead cells sedimented to the bottom of the tube.
2.3. Isotopic labeling of mouse lymphocytes and their detection by autoradiography Mouse thymocytes of AKR males 10 weeks of age were labeled with a single intraperitoneal injection (5 /aCi/g body weight) of [a H] thymidine (5 Ci/mM, Amersham-Searle, Arlington Heights, Ill.), four days before sacrifice (Matsuyama et al., 1966). This procedure labels over 90% of mouse thymocytes as detected by autoradiography. Slides with labeled thymocytes were prepared (see 2.8. below) and fixed in cold methanol and dried overnight at room temperature. They were then processed for autoradiography according to the procedure of Kopriwa and Leblond (1962). This method includes two 1 min stops in the following solutions: 100% ethanol; 95% ethanol; 80% ethanol, and distilled water. The slides were dried overnight and dipped into NBT-2 emulsion (Eastman Kodak & Co., Rochester, New York). They were dried for 30 min at 30°C. The slides were then stored in sealed plastic boxes at 4°C with an average exposure time of four weeks. They were developed in D-170 Kodak for 6 min and fixed with Kodak Fixer for 5 min. All processing was performed at 17°C in a light-proof room. After development, the slides were rinsed in tap water for 15 min, dried, and lightly stained with Giemsa.
Separation or fused lymphocytes
123
They were then passed through xylene for 1 hr and covered with coverslips mounted with 'Permount' (Fisher Scientific Co., Pittsburgh, Pa.).
2.4. Growth of Sendai virus, fusion procedure and culture conditions Sendai virus, obtained as a gift from J. Schwaber, was grown on the allantoi~ membranes of 9-day old embryonated chicken eggs. The eggs were inoculated with 0.2 ml of a 10-3 dilution of infected allantoic fluid as described by Klebe et al. (1970). After 72 hr incubation at 35°C, the eggs were placed at 4°C for 12 hr to induce clotting. The infected allantoic fluids were then harvested and virus was assayed by its capacity to agglutinate a suspension of 0.37% chicken red blood cells. The allantoic fluid was clarified by centrifugation at 2000 g for 20 min and virus was pelleted by centrifugation at 16,000g for 1 hr, after which the pellet was resuspended in a 0.5% BSA solution in PBS to 1/lOth the starting volume. After a second clarification, the virus suspension was inactivated with/3-propiolactone. An aqueous solution of fl-propiolactone was diluted to a 2% concentration with a buffered saline solution (100 ml of isotonic saline containing 1.68 g of Na2 CO3 and 0.5 ml of 4% Phenol Red). A virus suspension containing 10,000 hemagglutination units (HAU)/ml was brought to a final concentration of 0.05% /3-propiolactone with this solution. The virus suspension in the same solution was incubated at 4°C with shaking for 10 min and then at 37°C for 2 hr, and lastly, at 4°C overnight to completely inactive the ~3-propiolactone. The virus was diluted once again and stored in ampules at -70°C. For cell fusion, virus ( 2 0 0 - 5 0 0 HAU/2 × 106 cells) was incubated at 4°C with lymphocytes (107/ml) for 10 min in Dulbecco's medium without FCS. The cells were then pelleted by centrifugation at 100g for 10 min to increase cell contact. The pellet was warmed to 37°C for 10 min and then gently dispersed by pipetting. The cells were incubated for 2 hr in 5% CO2 at 37°C and then diluted to the final culture conditions: 2 × 106 cells in Dulbecco's complete medium with 10% FCS.
2.5. Velocity sedimentation Fused cells were partially purified by velocity sedimentation according to the method described by Miller and Philips (1969). Two identical chambers (12.5 cm diameter) were fitted with separate inflow and outflow ports and a common inflow channel for the FCS gradient (fig. 1). This arrangement allowed simultaneous study of two experimental groups under similar conditions. A separation started with the introduction of 30 ml of PBS into ea hch3~amber to minimize surface disturbances at the rising meniscus. The cell samples, in a 30 ml volume of PBS (6% FCS), were then introduced under the saline. This was followed by the slow introduction of 800ml of a linear 15-30% gradient of FCS in PBS. The filling rate was never greater than 40 ml/chamber/min and was lower for the first 150 ml (15 ml/min). The cells were allowed to settle from 2 to 4 hr. Fractions were taken off the
124
D.C. ALLISON, M. RALPH and E.P. COHEN GRADIENT INFLOW CHANNEL
STOPCOCK2
OUTFLOW CHANNEL 2
OUTFLOW CHANNEL 1
Fig. 1. Schematic diagram of velocity sedimentation apparatus. Experimental and control cells are introduced into separate sedimentation chambers through separate inflow channels. An anticonvection gradient (15-30% FCS in PBS) is slowly introduced underneath the cells which then are allowed to settle for 2 - 4 hr. Fractions are taken off the bottom of each chamber through the outflow channels. Larger cells, which have a greater sedimentation velocity, are in the earlier fractions. b o t t o m at a rate similar to the rate at which the chambers were filled. (The more rapidly sedimenting cells would be found in the earlier fractions.) The entire separation procedure was carried out at 4°C.
2.6. Cell preparation and media Thymus and spleen cells were pressed through a screen into cold PBS. Debris was allowed to settle for 10 min and the cells in the supernatant then pelleted in another tube. This pellet was resuspended in 'Tris buffered' NH4C1 (Boyle, 1968) for 7 min at 4°C (Shortman et al., 1972). The cells were then pelleted by centrifugation for 8 rain at 100g, washed twice in cold PBS, counted, and placed in the appropriate media. Cell culture was carried out in Dulbecco's Complete Medium (GIBCO, Grand Island, New York) supplemented with: L-glutamine (290mg/1), penicillin (100 units/ml), streptomycin (100/~g/ml), thymidine (10 -4 mM/ml), and 10% FCS. CF outbred mice were kindly provided by The University of Chicago Animal facilities. A K R and A/J mice were obtained from Jackson Laboratory, Bar Harbor, Maine.
2. 7. Preparation of cells for determination of radioactivity by scintillation counting Cells were radioactively labeled during a 2-hr incubation in Dulbecco's medium to which 5 mCi/ml [3 H] thymidine was added. After incubation they were centrifuged and the cell pellet was resuspended in 5% trichloroacetic acid at 4°C. This precipitate was caught on Whatman GF/C fiber filters. The filters were washed
Separation or fused lymphocytes
125
with 10 ml of cold methanol and vacuum-dried for 2 hr at 90°C after which they were placed in Liquifluor and the radioactivity in the filters was detected in a liquid scintillation counter. This method gave a linear correlation between cell number and radioactivity over a 7-fold dilution.
2.8. Preparation of cells for examination Cells were sedimented with the aid of a Cyto-Centrifuge (Shandon Southern Inst., Ltd., Camberly Surrey, England). Approximately 0.5 ml of cells (106/ml) were introduced into a centrifugation block and the cells then centrifuged for 5 min onto glass slides at 150g. The slides were fixed in cold methanol for 7 min, processed for autoradiography, and stained. Cells for the detection of 'streaming' were counted with a Coulter Counter (Coulter Electronics, Hialeah, Florida). All other cells counts were performed using a hemocytometer. Coded specimens were examined for frequency of binucleated and fused cells. A binucleated cell was considered to be a fused cell if it had at least 7 grains over one nucleus and none over the other. Statistical analysis was performed using the Chi Square method of analysis.
2.9. Mixed immune hemagglutination The procedure of Watkins and Grace (1967) was used. 'Sensitized Sheep Red Blood Cells' (SRBC) were prepared by incubating SRBC first with mouse antiSRBC antiserum and then rabbit anti-mouse IGG antiserum (Miles Lab., Kankakee, Ilk). Mixtures of AKR thymocytes and A/J spleen cells reacted with anti A/J antiserum from AKR mice. These cells were then incubated for one hour with the sensitized SRBC'. Slides were prepared in the usual manner.
3. RESULTS
3.1. Separation of viable cells by density gradients More than 90% of viable cells present in mixed cultures of thymocytes and spleen cells 2 4 - 7 2 hr after fusion were recovered in density gradients of BSA. Ninety-nine percent of these recovered cells excluded Trypan Blue. Fused cells were recovered by this method 72 hr after fusion (fig. 2).
3.2. Velocity sedimentation of thymocytes above and below the 'streaming limit' The 'streaming limit' is the cell concentration (3 × 106/ml) above which cells do not sediment independently. This cooperative streaming effect causes the cells to sediment rapidly. It is not known if size dependent sedimentation occurs under streaming conditions. To demonstrate streaming effects 5 × 109 thymocytes were
126
D.C. ALLISON, M. RALPH and E.P. COHEN
ill
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Fig. 2. A fused l y m p h o c y t e . [ 3 H ] t h y m i d i n e - l a b e l e d A K R t h y m o c y t e s were fused with nonlabeled A/J spleen cells. Fused cells in this population were purified by velocity sedimentation and dead cells eliminated from culture by density centrifugation. A binucleated cell with one labeled nucleus was considered fused if it showed 7 or more grains in the labeled nucleus. 100 !,JJ 9 0 80-
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SEDIMENTATION
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VELOCITY
4
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(mm/hr)
Fig. 3. D e m o n s t r a t i o n o f t h e 'streaming' p h e n o m e n o n . A 3-hr velocity sedimentation was performed with 5 X 109 CF t h y m o c y t e s (e) in one sedimentation chamber and 5 X 107 (A) in t h e other. The more rapid sedimentation o f t h e 5 X 109 cells is apparent.
Separation of fused lymphocytes
127
placed in one sedimentation chamber and 5 × 107 thymocytes in the other. A 3 hr sedimentation was performed under identical conditions for both chambers (fig. 3). The higher cell concentration exceeded the 'streaming limit' of the chamber by at least a factor of 50. The more rapid sedimentation of these cells was apparent.
3.3. Separation or fused lymphocytes above the streaming limit The streaming phenomenon would seem to limit the total number of cells that can be separated by velocity sedimentation. To determine if it was possible to partially purify fused cells at concentrations above which streaming takes place, [3 H] thymidine-labeled AKR thymocytes were mixed with non-labeled A/J spleen cells. One-half of the mixture was exposed to Sendai virus and the remainder of the mixture was treated in a similar manner except that Sendai virus was omitted. Approximately, 2 X 109 cells were placed in each sedimentation chamber and separation performed over a 3~ hr period. As controls, non-separated groups were kept under similar conditions. The most rapidly sedimentating cells (10% of the total)
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Fig. 4. Purification o f binucleated cells beneath the streaming limit. Sendai virus-treated CF t h y m o c y t e s purified above the streaming limit were purified a second time beneath the streaming limit. T h e peak o f binucleated cells (A) at 5.4 m m / h r occurred before t h e radioactively labeled single cell peak (e) and all other velocities studied (p < 0.01).
128
D.C. ALLISON, M. RALPH and E.P. COHEN
Table 1 Partial purification of binucleated and fused cells in a mixture of labeled thymocytes and nonlabeled spleen cells by velocity sedimentation above the streaming limit.
Sendai virus
Group 1
Group 2
Group 3
Group 4
-
+
-
+
Separation by velocity sedimentation
-
+
+
No. cells studied
3600
2322
4200
7400
No. and percentage of binucleated cells*
18 *0.50%)
12 (0.52%)
47 (1.12%)
85 (1.15%)
No. and percentage of fused cells**
0
1 (0.04%)
0
12 (0.16%)
* Labeling pattern of both nuclei identical. ** Binucleated cell with one unlabeled nucleus and the other with 7 or more autoradiographic grains.
were compared with these controls. After elimination of non-viable cells by BSA gradients, the cells were processed for the mixed immune hemagglutination reaction (Watkins and Grace, 1967). Autoradiographs of cells from the following groups were examined for the incidence o f binucleated and fused cells: 1) non-separated, non-virus-treated cells; 2) non-separated, virus-treated cells; 3) separated, non-virus-treated cells; and 4) separated, virus-treated cells. Fused cells as detected by autoradiography were found only in the Sendai virus-treated groups (p < 0.01, fig. 4) (table 1). Binucleated cells occurred in both Sendai- and non-treated groups, with only a slight increase in the Sendai treated-group. Separation o f the cells above the 'streaming limit' led to a 2 - 3 - f o l d increase in the incidence o f binucleated and fused cells (t9 < 0.01) (table 1).
3.4. Separation of binucleated cells beneath the streaming limit 3 H-Labeled CF1 thymocytes were treated with Sendai virus and put into culture for 12 hr. An initial separation was performed above the streaming limit. A study of the most rapidly sedimenting 12% o f the cells revealed a 2 - 3 - f o l d increase in binucleated cells (approximately 1.5/1000 to 3.7/1000). After elimination of nonviable cells by density gradient separation, this rapidly sedimentating fraction was further purified by velocity sedimentation at concentrations beneath the streaming limit (fig. 4). Several thousand cells were studied for the incidence of binucleated cells at selected velocities. A maximum percentage o f binucleated cells occurred at a sedimentation velocity o f 5.4 mm/hr. This peak was significantly higher than the other fractions studied and in the unseparated mixture (p < 0.01). It was also ahead
Separation or fused lymphocytes
129
of the sedimenting single cells measured by scintillation counting. The large amount of radioactivity in rapidly sedimenting fractions was due to the presence of residual cell clumps reflecting selection o f the initial separation.
4. DISCUSSION Velocity sedimentation is an effective way of enriching the proportion of lymphocytes in a mixture of fused and non-fused cells. Density separation with isoosmotic BSA may be used to eliminate non-viable cells and does not select against fused cells. The separation o f cells above the streaming limit may reflect: 1) a size dependent separation; 2) dilution of the cells by streaming through to a concentration beneath the streaming limit with size-dependent sedimentation following, or 3) an association of fused cells with rapidly sedimenting clumps. The last explanation is unlikely as the incidence of binucleated cells is increased in the separated, nonSendal-treated group (p < 0.01). Although much resolution is lost, separation above the streaming limit may be of some value when a low purification of a large number of fused cells is needed. Separation below the streaming limit is the obvious choice when experimental design calls for a high percentage of fused cells. In this work velocity sedimentation significantly lowered the time necessary to detect fused cells. This technique may also aid in the detection o f fused cell products in other systems. The demonstration of a high number of binucleated cells in non-Sendai-treated cultures is unexplained and emphasizes the necessity of having a labeling pattern, or other markers (Watkins and Grace, 1967; Frye a n d Edidin, 1970), to identify fused cells when morphological criteria are not sufficient.
ACKNOWLEDGEMENT This work was supported by a research grant from The Leukemia Research Foundation, Inc.
REFERENCES Boyle, W., 1968, Transplantation 6,761. Frye, L.D. and M. Edidin, 1970, J. Cell Sci. 7,319. Klebe, R.J., T-R. Chen and F.H. Ruddle, 1970, J. Cell Biol. 45, 74. Kopriwa, B.M. and C.P. Leblond, 1962, J. Histochem. and Cytochem. 10, 269. Matsuyama, M.B., M.N. Wiadrowski and D. Metcalf, 1966, J. Exptl. Med. 123,559. Miller, R.G. and R.A. Philips, 1969, J. Cell Physiol., 73, 191. Mosier, D.E. and C.W. Pierce, 1972, J. Exptl. Med. 136. Poste, G., 1970, Advan. Virus Res. 16,303. Shortman, K., N. Williams and P. Adams, 1972, J. Immunol. Methods 1,273. Watkins, J.F. and D.M. Grace, 1967, J. Cell Sci. 2, 193.