Cell Tissue Kinet. (1970) 3, 263-274.

ANTIBODY-PRODUCING CELLS: ANALYSIS A N D PURIFICATION BY VELOCITY

SEDIMENTATION

R . A. P H I L L I P S A N D R . G . M I L L E R

Department of Medical Biophysics, Unisersity of Toronto, and The Ontario Cancer Institute, Toronto, Ondario

(Receiiled 21 October 1969 ; rerision rereired 9 February 1970)

ABSTRACT

Velocity sedimentation cell separation is a simple and reproducible method for obtaining highly enriched populations of viable antibody-producing cells. Using suspensions of spleen cells prepared from mice immunized with sheep erythrocytes, fractions containing up to 2 % 19s-PFC and 25 % 7s-PFC can be obtained. Granu- locytes constitute almost all of the remaining cells in these fractions.

The sedimentation profile of 7s-PFC is very broad in comparison with that of cell populations known to be homogeneous in size (e.g. mouse erythrocytes). Analysis of the profile of 7s-PFC at different times after immunization suggests that the heterogeneity arises largely from the doubling in cell volume as a cell moves from one mitosis to the next. Early in the immune response, when the majority of the PFC are proliferating, the variation in sedimentation velocities is consistent with such a two-fold variation in cell volume. Late in the response, when most PFC have stopped proliferating, the sedimentation profile is more homogeneous. This analysis suggests that the fractionation procedure is sensitive enough to separate PFC according to their position in the cell cycle.

Sedimentation velocities were also measured for several other classes of cells found in spleen. Comparison of these values shows that sedimentation velocity is a useful parameter for characterizing different types of cells.

I N T R O D U C T I O N Studies on the differentiation of the cells of the immune system are complicated by the fact that they are usually located in tissues which are extremely heterogeneous in composition. For example, in the bone marrow or spleen of an adult mouse, the cells of the immune system are mixed together with large numbers of other types of cells from the hemopoietic system. Even in lymph nodes, cells making antibody of a particular specificity seldom

Correspondence: Dr R. A. Phillips, Ontario Cancer Institute, 500 Sherbourne Street, Toronto 5, Ontario, Canada.

263

264 R. A . Phillips and R. G . Miller represent more than 1 % of the total cells present. Progenitors of antibody-producing cells are present in even lower frequency (Kennedy et of., 1965). Experiments on the properties of antibody-producing cells or their progenitors have been severely limited by the lack of pure populations of the desired cells.

Several techniques of cell separation have been tested on lymphoid tissue (Brunette, McCulloch & Till, 1968; Plotz & Talal, 1967; Mage, Evans & Peterson, 1969; Wigzell & Anderson, 1969). While most separation techniques give some purification of antibody- producing cells, pure populations have not been achieved. However, even partial separations of cells on the basis of differences in their physical properties can be useful experimentally. For example, Haskill, Legge & Shortman (1969) have shown that density-gradient centri- fugation can be used to separate antibody-producing cells of different densities, and to investigate the properties of their progenitors.

Velocity sedimentation provides a simple method for separating cells, primarily on the basis of size, but to some degree also on the basis of density. This method has been used by Mage, Evans & Peterson, (1968) to separate antibody-producing cells from other cell types present in a spleen cell suspension. We have used a modification (Miller & Phillips, 1969) of their procedure for studies on antibody-producing cells. We find that velocity sedimentation yields populations of cells significantly enriched for antibody-producing cells and that the sedimentation profile can be used to analyse the proliferative states of antibody-producing cells. In addition, sedimentation velocity appears to be a good para- meter for characterizing functionally different cells.

M A T E R I A L S A N D M E T H O D S

Mice In all experiments the mice used were C3H/HenOci x C57BL/60ci FI hybrids, 6-8 weeks

old at the time for the first antigen dose. The mice were housed five to a cage and allowed food and water ad libitum.

Jmmutiizntion procedures atid assriys The antigen used in these studies was sheep erythrocytes (SRBC) (Woodlyn Farms,

Guelph, Ontario). Prior to use, either as antigen or as indicator in assays for plaque-forming cells, the SRBC were washed three times in phosphate-buffered saline (PBS) (Dulbecco & Vogt, 1954). When used as antigen, the sheep erythrocytes were diluted to give a concentra- tion of 8 x lo8 SRBC per ml and 0.5 ml of the suspension was injected intravenously into the tail veins of recipient mice.

The Jerne plaque assay was used to enumerate the number of cells making anti-SRBC antibody (Jerne, Nordin & Henry, 1963). The only modification of the original method was the use of CMRL 1066 (Parker, 1961) in place of Eagle’s medium. We have assayed for plaque-forming cells producing 7s antibody (7s-PFC) and for those producing 19s antibody (19s-PFC) by incubating the plates at 37°C with 10% guinea-pig serum plus or minus rabbit anti-mouse y-globulin antibody (Dresser & Wortis, 1965). Control experi- ments were routinely done to ensure that 19s-PFC were not suppressed in the indirect PFC assay and that the concentration of rabbit anti-mouse y-globulin antibody used developed the maximum number of plaques.

Antibody-producing cells 265 In primary immunization, the maximum number of 19s-PFC was found on day 4, at

which time about 0.1 % of the nucleated cells in the spleen of the mouse are making 19s antibody against sheep erythrocytes. In secondary immunization, for which a second intravenous injection of 4 x lo8 SRBC was given 4-6 weeks after the first, maximum num- bers of both 7S- and 19s-PFC were found on day 4, at which time about 1 % and 0.1 % of the nucleated cells in the spleen were making 7 s and 19s antibody, respectively.

Preparution of cell suspensions Spleen cells from immunized mice were used in all cell separation experiments. To

prepare a single cell suspension, the spleens were removed, placed in cold PBS, and cut into small pieces with scissors. These small pieces were then aspirated in and out of a disposable plastic syringe until the large pieces of debris were colorless. The debris was removed and the remaining suspension was filtered through a 60 mesh stainless steel screen into a glass centrifuge tube which was spun a t 250 g for 10 min in a refrigerated centrifuge. The supernatant was discarded and the pellet of cells resuspended in cold PBS. In recent experiments we have found that small clumps of cells can be removed a t this stage if the cells are filtered through a capillary array filter (Mosaic Fabrications, Sturbridge, Mass.) with a pore size of 35 p.

Cell sepuration Velocity sedimentation was used to separate spleen cell suspensions on the basis of cell

size. The method used was identical to the technique described by Miller & Phillips (1969) which is similar to a method previously described by Peterson & Evans (1967). In brief, a suspension of cells in PBS plus 3 % fetal calf serum is placed on top of a non-linear gradient of 5-30?; fetal calf serum in PBS. The cells are allowed to sediment through the gradient for 3-4 hr and fractions are collected from the bottom of the gradient. Fractions of 15 ml were collected in all experiments except the one shown in Fig. 2 where 6-ml fractions were collected in an attempt to improve resolution. Seventy to 90 % of the input cells are recovered from the gradient. Similar recoveries were obtained for all individual cell types examined; selective loss of one or more types of cells was not observed.

All cell counts on the starting spleen cell suspensions were done with a hemocytometer. Cell counts for the various fractions of the gradient were done with a Coulter counter. When necessary, saponin was added to obtain the nucleated cell count. In some experi- ments fractions from the bottom of the gradient were also counted with a specially designed, large hemocytometer because the concentrations of cells were below the background counts in the electronic counter.

Grunulocytes Granulocytes were enumerated using the peroxidase assay described by Fowler et UI.

(1967). In this technique cells are collected on Millipore filters, stained first with benzidine to detect peroxidase activity and then with Harris stain to color the nuclei.

R E S U L T S Sedimentation projles of spleen cells f rom immunized mice

Antibody is synthesized predominantly by large plasma cells, which, since they are larger than most other cells in spleen tissue, should sediment much more rapidly than the majority

18

266 R. A . Phillips and R. G. Miller of the spleen cells. Fig. 1 shows a typical velocity sedimentation distribution of spleen cells obtained from a mouse 4 days after primary immunization with SRBC. The distributions of nucleated cells, erythrocytes and 19s-PFC are indicated. The data are plotted on a log- arithmic scale in order to emphasize small but reproducible features of the sedimentation profile. Apart from the PFC, four major features dominate the profile: the erythrocytes, which sediment in a narrow band at a rate of 2.0 mm/hr; two bands of nucleated cells,

Bottom Fraction number TOP

FIG. I . Sedimentation profile of spleen cells from a mouse 4 days after primary immunization. The hatched bar indicates the initial position of the cell band. Fractions are numbered in the order of collection from the bottom of the chamber. The circles indicate nucleated cells/ml of gradient. The solid line represents the total cell count. The dashed curve (peaking in fractions 15-16) represents erythrocytes/ml. The triangles are numbers of 19S-PFClml.

one at 1.9 mm/hr and the other at 2.9 mm/hr; and a peak of small particles just beneath the starting band, seen only with the electronic cell counter. Microscopic observa- tion of fractions in this last region show no intact cells, and we interpret this peak to be cellular debris. In addition, it appears that the peak of slowly sedimenting nucleated cells (1.9 mm/hr) represent damaged cells. These cells do not incorporate vital dyes nor has any function been associated with them. Variations in the number of cells in this peak have been correlated with the force required to prepare the cell suspension.

Antibody-producing cells 267 As expected, 19s-PFCs have a relatively large peak sedimentation velocity or ‘s’ value

(4-7 mm/hr) indicating that 19s-PFC are larger than most of the other cells in mouse spleen. In comparison to the distributions of erythrocytes and nucleated cells, the distribution of PFC is much broader, indicating a heterogeneous population of cells. This observation will be considered in detail in the following section. A small number of plaques are also found beneath the debris peak. These plaques, which are not associated with intact cells, were observed and studied by Mage el al. (1968). ‘False’ plaques associated with cell debris and fibrin clots have also been described by Roseman, Rowley & Fitch (1968).

To compare the sedimentation profiles of cells making 19s and 7s antibody, spleen cells were taken from mice 4 days following a second immunization with SRBC. The spleen cell sedimentation profile, along with the distribution of 7s- and 19s-PFC and peroxidase

10 20 30 40 50 60 )ttom TOP Ffactlon number

FIG. 2. Sedimentation profile of spleen cells from a mouse 4 days after secondary immunization. The distributions of nucleated cells (solid line), erythrocytes (dashed line) granulocytes (0), 7s-PFC (A) and 19s-PFC (0) are indicated. The center of the input band is off scale in fraction 65.

positive cells, is shown in Fig. 2. The distributions of erythrocytes and nucleated cells are similar to those shown in Fig. 1, although the smaller fractions used in this experiment give slightly better resolution of the 1.9 and 2.9 mm/hr nucleated cell peaks. The major difference from the distribution shown in Fig. 1 is the presence in Fig. 2 of a large shoulder in the distribution of nucleated cells in fractions 25-30 (s value of about 4.5 mm/hr). We often see this shoulder in spleen cell suspensions after secondary immunization with SRBC and,

268 R. A . Phillips and R. G. Miller as can be seen from the functional assays, it corresponds to the peak positions of both PFC and granulocytes.

The distributions of 7S- and 19s-PFC are similar to each other and to the distribution shown in Fig. 1. Most (88%) of the 7S-PFC are found in fractions 1-30. These fractions contain only 8.2% of the nucleated cells. In the region of fraction 10, up to 10% of the cells are 7S-PFC compared with 0.3% in the input suspension. For comparison, Mage et a/. (1968) obtained 18-fold enrichment and 1 % PFC in their most enriched fractions; however, their separation were performed 1 day before the time of the peak response.

k

The distribution of 19S-producing cells is similar in suspensions obtained either from singly or doubly immunized mice. In both cases, there is roughly a 20- to 40-fold increase in the frequency of PFC in the fractions containing the largest cells. In unfractionated suspensions 19S-PFC are usually in a frequency of O.OS-O.l%; near the bottom of the gradient the frequency of 19s-PFC increases to a maximum of 1-2%.

For this separation technique to be useful experimentally, it is essential that it be repro- ducible. We have made ten measurements of the 7S-PFC profile of spleen cells from mice 4 days after secondary immunization. Fig. 3 gives the 7S-PFC distribution (plotted against sedimentation velocity) obtained in four of these experiments and Table 1 lists parameters

Ailtibody-producing cells 269 from all ten experiments. Here we have calculated the sedimentation velocity and the width of the PFC peak in all ten experiments. The mean s value found here for 7s-PFC (4.4 i 0.3 mmjhr) is slightly smaller than that for 19s-PFC (4.7 ~t 0.2 mm/hr). These measurements show that the separation procedure is highly reproducible. A similar high degree of repro- ducibility has been observed by Brubaker & Evans (1969) in their sedimentation analysis.

Table 1 also lists both the maximum percentage of 7s-PFC and the maximum enrichment over control for all ten experiments. The maximum percentage of 7s-PFC approaches an upper limit of 25-30% and appears to be correlated only poorly with enrichment over control which varies from 14-fold to 110-fold. We consider the maximum percentage

TABLE I . Reproducibility of the 7s-PFC separation

Sedimentation velocities (mmihr) 7s-PFC purification Exp. No. RBC 7s-PFC Peak width* Per cent? Enrichment$

1 2 3 4 5 6 7 8 9s:

10

2.0 1.9 2.0 1.9 2. I 2.0 2. I 2.0 1.8 2.0

4.2 4.5 4.1 4. I 4.5 4.4 4.7 4.8 3.9 4.6

1.4 1.6 1.2 1.9 1.4 1.6 I .6 1.7 1.5 1.5

8 28 25 10 13 4

I 1 5

11 25

- 22 38 14

110 29 22 41 15 18 62

Mean f SD 2.0 * 0.1 4.4 i 0.3 1.5 & 0.2 1 4 & 8 37 i 28

* Peak width is defined as the difference in sedimentation velocities between the two fractions that have

t The value shown is the maximum obtained in the particular experiment. $ Enrichment is defined as the maximum per cent PFC divided by the per cent PFC in theinitial suspension. $ In this experiment the sedimentation velocities of both RBC and 7s-PFC are low. Whenever abnormal

half as many PFC/ml as the peak.

sedimentation velocities are encountered, all types of cells are affected in the same proportion.

obtained to be a more practical measure of the purification achieved. We do not under- stand the wide variations in enrichment. I t might result from variations in the immune response in our mice.

In connection with purification of 7s-PFC it is important to note that granulocytes are the major cell type in the region most enriched for 7s-PFC. The granulocyte distribution has two peaks (Fig. 2); in the major peak SO-SO% of the cells in each fraction are granulo- cytes compared to only 5 % in the starting suspension. Thus, in this region of the gradient almost all of the cells present can be classified either as granulocytes or antibody-producing cells. Preliminary experiments indicate that removal of granulocytes from the starting suspension gives significant enrichment of plasma cells in fractions near the bottom of the gradient.

Heterogetieity of the PFC distributiori: cell cycle model All of the PFC distributions presented have a large dispersion in sedimentation velocity,

much larger than if the cells making antibody all had the same size and density. One

270 R. A . Phillips and R. G . Miller possibility is that several morphologically distinct populations of cells are making antibody. However, the distributions we observe look more like a continuous distribution of a single cell type, and we have assumed this to be the case. Cell proliferation is known to be involved in the immune response (Syeklocha et al., 1966; Tanneberg & Malaviya, 1968; Makela & Nossal, 1962; Krisch, 1969), and is one factor that can cause size heterogeneity in an otherwise homogeneous population. Cells immediately before mitosis have twice the volume of cells immediately after mitosis. Because the sedimentation velocity of cells is

Froction number Bottom

Fic. 4. Sedimentation profiles of 7s-PFC from spleen on day 3 (0) and day 7 (A) after secondary immunization. The dashed line represents the theoretical profile calculated for a proliferating population of cells of constant density. See text.

directly proportional to ( ~ o l u m e ) ~ ’ ~ , the sedimentation velocities of pre- and post-mitotic cells (assuming constant cell density) should be related as follows :

u2 = 22’3~1, = 1.590,

where 2’2 and 11, are the sedimentation velocities of pre- and post-mitotic cells, respectively. In addition, because the mitotic process produces two small cells from one large cell, in a rapidly proliferating population there should be twice as many post-mitotic as pre-mitotic cells.

In Fig. 2, the arrow designated u 1 was drawn at 50 %of the PFC peak to represent PFC that we assume have just passed through mitosis. Using this sedimentation velocity we calculated the sedimentation velocity zi2 shown on the figure. In this particular experiment, 81 % of the PFC are included in the fractions between the two arrows, indicating that the dispersion

At1 tibody-producing cells 27 1

of the PFC population could indeed arise principally from the proliferation of a homo- geneous population of cells of approximately constant density.

A more definitive test of the hypothesis is to compare the size distribution of a rapidly proliferating population of PFC with the distribution obtained from a population of PFC that is not proliferating. Three days after a second immunization, most antibody-prodlicing cells are in a state of rapid proliferation, but 7 days after immunization most antibody- producing cells have matured and are no longer dividing (Makela & Nossal, 1962; Krisch, 1969). Fig. 4 shows the distributions of 7s-PFC from spleen for these two cases. T o facilitate comparison of the distributions, the data have been plotted as a fraction of the total PFC recovered from the gradient. The PFC distribution obtained 3 days after immunization resembles remarkably well the idealized distribution (dashed line) expected for a cycling population. In contrast, the distribution of PFC obtained 7 days after immunization is much more homogeneous and at a position consistent with the view that mature cells go out of cycle shortly following mitosis (Lajtha, 1963). The detailed shape of the day 3 curve and the way the day 7 curve differs from it strongly support the view that the broad distri- butions of PFC seen in velocity sedimentation separation are a result of the volume change associated with cell proliferation in a homogeneous population of cells. Comparison of the two curves in Fig. 4 indicate that the PFC distributions obtained 4 days after immunization are likely to be a mixture of mature, non-proliferating cells and immature, proliferating cells. Thus, the distributions of Figs. 2 and 3, which have a distinct peak and a shoulder toward the large, rapidly sedimenting cells, likely represent a mixture of mature, non- dividing PFC and immature, proliferating PFC.

D I S C U S S I O N Our conclusion that antibody-producing cells are a relatively homogeneous population of cells differing in volume mainly as a result of proliferation should be compared to two observations indicating additional sources of heterogeneity. First, Haskill et al. (1969) measured the density distribution of antibody-producing cells and found a wide dispersion of cell densities. I f the densities they measured represent actual cell densities, we would have expected even greater heterogeneity in sedimenation velocity, since this parameter depends to some extent on cell density. In an attempt to resolve this problem, we are currently measuring the density distribution of PFC to determine the contribution of density heterogeneity to heterogeneity in sedimentation velocity. Second, Buell & Fahey (1969) reported that cells in tissue culture synthesize significant quantities of antibody only during late G , phase and early S phase. If our interpretation of the sedimentation profile of PFC is correct, velocity sedimentation separates cells according to their phase in the cell cycle. Thus, our data indicate that cells in all portions of the cell cycle actively synthesize antibody. The differences between the two sets of data could be in the source of cells used in the experiments. We used normal mouse spleen cells obtained after immunization itz i:iuo. Buell & Fahey (1969) used human lymphoma cells growing in uitro; these cells in culture synthesize various classes of antibody molecules of unknown specificity and may not provide a good model for studying antibody synthesis.

Several methods have been tried by other investigators to achieve purified populations of antibody-producing cells, but with the exception of the method described by Makinodan et nl. (1967) the purifications achieved have been minimal. These workers obtained highly

272 R. A . Phillips and R. G. Miller purified populations of antibody-producing cells by placing heavily irradiated spleen cell suspensions from immunized mice in diffusion chambers and implanting these chambers into the peritoneal cavities of irradiated recipients. Although this technique gives a large proportion of plasma cells after 5 days of incubation, the use of radiation makes it unsuitable for studies that require proliferation of the purified cells. The advantages of velocity sedimentation over the other methods is that it is possible to obtain in high yield populations either enriched or depleted for antibody-producing cells without any concomitent loss in cell viability.

Sedimentation velocity may provide a useful parameter to characterize quantitatively a particular cell type. The numerical values found for the sedimentation velocity (s value) of a particular cell type are remarkably reproducible from one experiment to the next. The s value and a small set of similarly reproducible physical quantities (e.g. density, volume, electrophoretic mobility) may better characterize a particular cell type than the generally used morphological criteria. The major advantage of such a characterization is that it would be quantitative rather than qualitative. In addition, such a characterization of cells would be experimentally useful in that these parameters would suggest methods for achieving separation and purification of particular cell types. We are currently working along several lines to see if such a characterization is feasible. The data of Table 2 represent a start in this direction. Here we show the s values for the seven classes of cells mentioned

TABLE 2. Sedimentation velocity of cells found in mouse spleen

No. of Sedimentation velo- Type of cell measurements city (Mean SD)

Erythrocyte 10 2.0 i 0.1 mm/hr Nucleated cells-slow 7 1.9 i 0.1 mm/hr Nucleated cells-fast 7 2.9 & 0.2 mm/hr Granulocy tes-slow 5 4.3 f 0.2 mm/hr Granulocytes-fast 5 5.9 i 0.3 nim/hr 19s-PFC 5 4.7 i 0-2 mm/hr 7s-PFC 10 4.4 j: 0.3 mm/hr

in this paper (all of these peaks are shown in Fig. 2). Although each type of cell can be characterized by a unique s value, i t must be emphasized that these values are strongly dependent on the conditions of sedimentation. Changes in the viscosity, density, or tonicity of the supporting medium will alter the s value. Thus, considerable care must be taken to ensure the constancy of conditions from one experiment to another.

The data presented here have suggested to us two lines of investigation that utilize velocity sedimentation cell separation as a tool in helping to understand the cellular events of the immune system. First, one can obtain a pure enough population of antibody-produ- cing cells to examine these cells directly for chromosome markers and thus uniquely determine their origin (Edwards, Miller & Phillips, 1970). Second, one can locate the precursors of antibody-producing cells on the gradient and follow them through the gradient during the initiation and development of the immune response (Miller & Phillips, 1970). We are currently pursuing both objectives.

Antibody-producing cells 273

A C K N O W L E D G M E N T S

We wish to thank Mr B. Kuba, Mr M. Kerr and Miss H. Mooney for their competent technical assistance.

This work was supported by the National Cancer Institute of Canada, the Medical Research Council (Grant MA-3017), and the Donner Canadian Foundation.

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