Immunologic Methods for the Identification of Cell Types. I. Specific Antibodies That Distinguish Between Mammary Gland Epithelial Cells and Fibroblasts 1 , 2 Karen Thompson, Roberto L. Ceriani, Donald Wong, and S. Abraham 3, 4 ABSTRACT-Antibodies were produced against intact mouse mammary epithelial cells and cleared fat pad fibroblasts; after appropriate absorption, two specific antibody preparations were obtained. The antimammary epithelial cell preparation did not appear to be strain- or species-specific. The antimammary fibroblast preparation recognized both mammary and fetal fibroblasts. These findings suggested that each cell type possessed distinct immunogenic components. These components were characteristic of each cell type and could be used to identify the respective cells by an immunofluorescence technique. Characterization of the antigenic component of mouse mammary epithelial cells demonstrated that this antigen was released during enzymatic cell dissociation and was regenerated under in vitro culture.-J Natl Cancer Inst 57: 167-172, 1976.

Identification of cells in culture has been one of the most vexing problems facing the cell biologist. Methods that depend upon cell morphology have not been entirely satisfactory (l, 2), because cells in culture take on a different appearance from that seen in the tissue of ~:nigin. Techniques that take advantage of the cells' abilIty to produce specific products as part of a tissue or gland have also been unsuccessful. Specifically, mammary gland epithelial cells lose their special ability to synthesize the compounds characteristic of lactation (37) soon after culture. Whether this is because of an actual loss in function due to the different hormone n:ilieu, specific culture conditions, or an overgrowth by different cell types will remain unanswered until cell types grown in culture can be identified with absolute certainty. To resolve this problem, we have concentrated our efforts on the development of cell markers that do not depend upon cell morphology or function. Much evidence supports the view that surface components of cells contain specific antigens (8-13). It is possible, therefore, to develop markers that use these sites. Here we report the production of two cell-specific antibodies, one against MMEC and the other against MMFC. Use of these antibody preparations permits a clear identification of such cells in culture. Anti-MMEC w.ere developed in rabbits and anti-MMFC in guinea pigs; both were visualized in the fluorescence microscope with the use of a fluorescein-labeled goat antiserum. against y-globulin. Details of this procedure, with pertment data on characterization of the antigens on the MMEC, are reported here. MATERIALS AND METHODS Animals and Tissue Sources

All mice were of the BALB/c strain unless otherwise noted. Mammary tissues were obtained from virgin (60~O da~s old), 14-16-day pregnant, and 1O-12-day lactatIng mICe. In addition, the glands of 3-week-old female mice were surgically cleared of the glandular elements by the method of DeOme et al. (14) and the cleared fat VOL. 57, NO. I, JULY 1976

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pads taken when the mice were 90-100 days of age. Sprague-Dawley rats, when used, were in their 14-16th day of pregnancy. The New Zealand White adult rabbits (2-3 kg) used for production of antibodies were obtained from Big Pine Rabbitry, Saratoga, California; the guinea pigs were from Diablo Laboratories, Berkeley, California. All other animals were bred in our vivarium. Fibroblasts were isolated from two tissue sources: the cleared mammary gland fat pads, and fetuses from the pregnant mice used in this study. Cell Dissociation Techniques

After the animals were killed by cervical dislocation, the d.e~ired tissues were quickly removed under aseptic conditIOns. Lymph nodes and muscular tissues were removed from mammary glands. All tissues were cut with scalpels into pieces 1-2 mm 3 and washed with CMF, pH 7.4 (15). All solutions used with tissues or media used for culture contained 3.5 mg penicillin G and 5 mg streptomycin/lOO ml. Fetuses were trimmed of eyes and abdominal contents and then cut into small pieces. Each tissue was individually subjected to one of the following procedures for cell dispersion: Method A.-Digestion was performed with 0.25% trypsin (1 :250 Difco certified; Difco Laboratories, Inc., Detroit, Mich.) plus 0.02% EDT A in CMF for 20 minutes (10 ml/g tissue) at 37° C with gyratory agitation. After the agitation was stopped, the tissue pieces were allowed to settle and the supernatant fraction was removed. This fraction was centrifuged at 150 X g for 5 minutes to sediment the cells, and digestion of the pieces was repeated twice, each time with fresh enzyme solution. The cell pellets were combined and resuspended in fresh Waymouth's medium (MB 752/1; Grand Island Biological Co., Santa Clara, Calif.) containing 20% FCS and finally passed through a 10- f.Jnylon mesh Nitex filter (Tobler, Ernst, and Traber, Elmsford, N.Y.). Samples of the single-cell suspension ABBREVIATIONS USED: MMEC=mouse mammary gland epithelial cells; MMFC=mouse fibroblasts; anti-MMEC=antibodies against intact mouse mammary gland epithelial cells; anti-MMFC=antibodies agail?st intact mouse fibroblasts; CMF=calcium- and magnesium-free medIUm; FCS=fetal calf serum; M-RBC=mouse red blood cells; FIGaRyG=f1uorescent goat antiserum against rabbit y-globulin; FIGaGPyG.=f1uorescent goat antiserum against guinea pig y-globulin; BSA=bovme serum albumin; cpm=counts per minute; MSA=mouse serum albumin; PBS=phosphate-buffered saline. I Received October 16, 1975; accepted January 16, 1976. 2 Supported by Public Health Service contract NOI CB33906 from the Division of Cancer Biology and Diagnosis, National Cancer institute. 3 ~ruce Lyon Memorial Research Laboratory, Children's Hospital MedIcal Center, 51st and Grove Streets, Oakland, Calif. 94609. 4 We thank Drs. J. Bartley, C. Henry, R. Moretti, and S. Smith for helpful discussions in preparing this manuscript and J. Hardesty for typmg.

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were then assayed for cell numbers in a hemacytometer. Method B.-Digestion with collagenase was performed as previously described (16). The final cell suspension was filtered through the 1O-1L N itex filter, washed twice in Waymouth's medium, and counted for cell number. Method C.-Cell dispersion was essentially obtained by the method of Weipjes and Prop (15), except that a 1O-1L Nitex filter was used instead of sintered glass filters. Culture Techniques

Suspension cultures were initiated with 1 x 106 cells/ml and maintained in sealed Erlenmeyer flasks containing 10% FCS in Waymouth's medium. They were continuously rotated in a gyratory agitator at 37° C for various intervals in an atmosphere of 95% O 2 and 5% CO 2 , At the end of the culture period, the suspended cells were collected by centrifugation and washed twice with Waymouth's medium. Monolayer cultures were grown in plastic petri dishes (Falcon Plastics, Inc., Cockeysville, Md.). Epithelial cells were seeded at a density of 3 X 105 /cm 2 , and fibroblasts at a density of 1 x 105 /cm 2 • The epithelial cells and fibroblasts were maintained at 37° C in an atmosphere of95% air and 5% CO 2 in Waymouth's medium containing 20 and 10% FCS, respectively, for the times indicated in the tables. In some instances, the cells were removed from the plate by incubation with a solution composed of 0.25% trypsin in CMF at 37° C. When required, cells were also removed at room temperature with a rubber policeman. Preparation and Purification of Antibodies

Anti-MMEC.-MMEC obtained from pregnant mice by method C were grown in suspension culture for 48 hours, washed two times with Waymouth's medium without FCS, and used for the production of antibodies as follows: New Zealand White rabbits were given 3 weekly ip injections of 20x 106 MMEC in 1 ml Waymouth's medium. The rabbits were bled 1 week after the last injection to test for antibody titers by an indirect fluorescence assay (17). They were subsequently bled weekly. A similar number of cultured MMEC were injected every 2 weeks to maintain antibody titers. A 40% saturated ammonium sulfate dialyzed y-globulin fraction of the pooled sera was prepared and then absorbed at room temperature for 3 hours with 113 volume of washed, packed M-RBC. This absorption was sufficient to produce no detectable fluorescent staining of M-RBC. For removal of fibroblast-specific antibodies, 1 ml y-globulin (M-RBC absorbed) was absorbed with 20x 106 fibroblasts. We found that fibroblasts obtained from either monolayer-cultured cleared mammary fat pads or monolayer-cultured fetal fibroblasts were essentially equal in eliminating the staining of cultured fibroblasts. Following this absorption, the final y-globulin preparation (anti-MMEC) produced a greater than 80% positive staining of the suspension-cultured MMEC (c[. Dl and D2 with Cl and C2, figs. 1,2). This anti-MMEC J

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preparation (12-15 mg protein/ml) was frozen in 0.2-ml aliquots. Anti-MMFC.-Fibroblasts from cleared fat pads obtained by method A were cultured as monolayers to confluency, which usually takes 10 days. Cells were removed with a rubber policeman and washed two times in Way mouth's medium; finally, 5x 106 cells were resuspended in I ml Waymouth's medium. This suspension was injected ip into guinea pigs weekly, 1 ml per animal, by a schedule similar to that for the rabbits given above. One month after the initial injection, the serum was isolated and its antibody titer was tested by an indirect fluorescence assay (17). A y-globulin fraction was obtained and first absorbed with M-RBC, then with 50X 106 MMEC/ml as given above. The final purified preparation (anti-MMFC) was frozen in 0.2-ml samples (12-15 mg protein/ml). Identification of Cells by Immunofluorescence

Fluorescence assays of cells bearing antigens were performed with either FIGaRyG or FIGaGP YG (both obtained from Antibodies, Inc., Davis, Calif.) in an indirect two-step technique. In the first step, an aliquot of 25 ILl antibody was added to an equal volume of a cell suspension containing approximately 8-16x 107 cells/ml in Waymouth's medium with 0.5% BSA (35% solution; Pentex Biochemicals, Kankakee, Ill.) and 10- 2 M sodium azide. The mixture was then incubated at 37° C for 20 minutes and washed twice at room temperature with the same medium. The second amplifying layer was added by incubation with 25 ILl of the fluorescent labeled goat antiserum against y-globulin (20 mg protein/ml) for 20 minutes at 37° C. After two washes with the medium, the cells were resuspended in 50-100 ILl medium. Cells were examined within 18 hours after staining by the wetmount technique with a Leitz Dialux microscope equipped for fluorescent epi-illumination (100- W tungsten halogen lamp) with one KP500 excitation filter and one K530 barrier filter. For phase contrast viewing, we used a 15-W tungsten lamp. ["H) Fucose Incorporation

Several pieces of mammary glands (=1-2 mm 3 ; 100 mg) or freshly dissociated MMEC (method C; 1 x HF cells), both obtained from pregnant BALB/c mice, were incubated with 5 ml Waymouth's medium containing 10% FCS and 10 ILCi [3H]fucose for varying periods of time at 37° C, 70 turns per minute, with 95% O 2 and 5% CO 2 as gas phase. Samples of the labeled cells were removed after 0, 16, 18, 24, 48, and 72 hours, whereas tissue was removed at 48 hours; both preparations were washed in Way mouth's medium with 10% FCS. At each time interval in some experiments, the cell samples were divided into three parts. One part was used for the assay of tritium incorporation into 5% trichloroacetic acidprecipitable and acetone-insoluble (three washes) material, another for the determination of DNA (18), and the third for estimation of the degree of MMEC antibody binding. The indirect immunofluorescence technique VOL. 57, NO.1, JULY 1976

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(described above) was used. [3H]fucose incorporation was measured in terms of 3H cpm/ ILg DNA. Further treatment of the tissue pieces and cells is outlined in the tables.

TABLE

l.-Cell yield: Comparison of methods used for cell dissociation a

Physiologic state (and No.) of animals

Dissociation method 10-6 cells/g wet wt of gland

Affinity Chromatography

Two affinity immunoabsorbents were prepared: one with rabbit anti-MMEC and the other with rabbit antiMSA (fraction V; Pentex Biochemicals). The stationary matrix, Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, N.J.), was activated with cyanogen bromide and then coupled to each antibody preparation (19). Columns (1 x5 cm) were prepared with each affinity immunoabsorbent and equilibrated with PBS, pH 7.3. A 2-ml sample was added to the column and allowed to react for 1 hour at room temperature. The column was then washed with four column volumes of PBS, which served to remove all unabsorbed material. Specifically bound antigen material was eluted with 30-40 ml of 1 M acetic acid. Fractions (0.5 ml) were collected and aliquots were assayed for radioactivity. RESULTS

As a result of our studies with the three different techniques of cell dissociation, we adopted method C for MMEC cell isolation (table 1). This method produced six to seven times more cells from tissue of mid pregnant and lactating mice than did method B. Although larger numbers of cells can be obtained from mammary glands taken from lactating mice, those from pregnant animals were preferred, inasmuch as the cells obtained from pregnant mice gave a greater plating efficiency in monolayer cultures and a larger percentage of live cells after 48 hours of suspension cultures. Specificity of Anti-MMEC

Studies with lymphocytes (20) have shown that certain antigens stained with multivalent ligands do not remain uniformly distributed on the cell surface. The extent of redistribution of surface antigens was found to be temperature-dependent; for this reason we investigated the fluorescent staining of MMEC after treatment with antiMMEC at various temperatures. Cells cultured in suspension for 48 hours were subjected to our two-step procedure at 4, 20, and 37° C. We did not observe any redistribution; a diffuse type of fluorescence was observed at all temperatures, with a greater intensity seen at the cell outline (figs. 1,2; Cl, C2). The specificity of the anti-MMEC is demonstrated in table 2. Anti-MMEC specifically bound to MMEC but did not bind to cells from spleen, brain, liver, kidney, MMEC freshly dispersed enzymatically, and non cultured MMEC. Fibroblasts isolated from either fetal tissues or cleared fat pads did not bind (figs. 1, 2; D 1 and D2). Further, the anti-MMEC did not appear to be either strain- or species-specific (data not shown). Dispersed and cultured MMEC obtained from DBA, C3Hf, C57BL, A, and GR/A pregnant mice and pregnant Sprague-Dawley rats bound specifically anti-MMEC (alVOL. 57, NO.

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Virgin (1) Midpregnant (3) Lactating (3)

0.6 4.8 6.2

0.4 2.3 2.1

1

24 41

Tissues were taken from individual BALB/c mice and the cells dissociated according to the methods described in the text. Each digestion flask contained 1 g tissue/5 ml enzyme solution. Values are averages. b Trypsin. C Collagenase. d Collagenase plus hyaluronidase plus pronase. a

TABLE

2.-Tissue specificity of anti-BALB/c MMEC a Dissociation method b

Cells M-RBC Spleen Brain Liver Kidney Fetal fibroblasts

None Mechanical Mechanical Mechanical Mechanical A

Fat pad fibroblasts

A

MMEC (midpregnant) MMEC (midpregnant)

C C

MMEC (mid pregnant) MMEC (midpregnant)

Mechanical C

a

Culture No No No No No Monolayer, 14 daysd Monolayer, 14 daysd No Suspension, 2 days No Monolayer, 10 daysd

Fluorescence intensityc 0 0 0 0 0 0 0 0

4+ 3+ 3+

Mammary cells, MMEC, and MMFC were isolated as described

in text. Other tissues taken from virgin BALB/c mice were minced

and the cells released by a mechanical method that involved the manual manipulation of a loose-fitting tissue grinder (Teflon pestle, glass barrell with 20 volumes of Waymouth's medium. The tissue pieces were allowed to settle and the supernatant was centrifuged at 150xg for 5 minutes to sediment the cells. The resuspended cells (8x 107 cells/mll were tested for antibody binding (see text). b A=trypsin; C=collagenase plus hyaluronidase plus pronase. c Intensity was judged on a scale of 0 to 4+ (most intense). d Cells were removed with a rubber policeman after incubation.

though at different levels). In addition, MMEC obtained during different physiologic states of the BALB/c mammary gland (from virgin, pregnant, and lactating animals), bound anti-MMEC and FlGaRy(; equally. Only MMEC that had been cultured in suspension (figs. 1,2; c[. Bl and B2 with Cl and C2) or obtained by mechanical teasing from monolayers or tissues bound to the antibody (table 2). Two types of controls suggested the lack of nonspecific binding. The first, in which normal rabbit y-globulin and FlGaRyG were reacted with the MMEC, showed a negative reaction (figs. 1, 2; Al and A2). The second, in which the anti-MMEC was omitted from the two-stage staining procedure, was also always negative. In addition, we showed that freshly dissociated MMEC did not bind the anti-MMEC (figs. 1, 2; c[. Bl and B2 with Cl and C2). Such findings demonstrated that cells freshly dis soJ

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ciated by treatments with enzymes have lost their antigenic components, but that these components can be restored during culture. Indeed, MMEC cultured in suspension began to show anti-MMEC binding after 18 hours. This ability increased up to 48 hours, when fluorescence was maximum with the two-step indirect technique. In these experiments, we monitored regeneration of cell surface material by the incorporation of [3H]fucose into cell glycoproteins (table 3). Apparently, 48-72 hours were required for these cells to fully recover from the effects of the dissociation by enzymes. Both the percentage of cells binding anti-MMEC and the intensity of fluorescent staining reached maximum values after that time. When cells were grown for as long as 14 days, their ability to bind the anti-MMEC was undiminished. Experiments were designed to investigate these regenerable components. Tissue slices obtained from midpregnant mouse mammary glands were labeled with [3H]fucose by incubation for 48 hours (as in "Materials and Methods"). At the end of that time, the tissue was treated with collagenase and hyaluronidase (first part of method C) and the cells were tested for binding with the anti-MMEC. The results given in table 4 demonstrate that after removal of 26% of the [3H]fucose taken up by the tissue, the resulting intact cells no longer bound the antibody. It is clear that this treatment with enzymes removed the antigen substances from the cells. Once again, when these cells were cultured in suspension for 48 hours, the ability to bind anti-MMEC was restored (table 4, step 4). To further analyze this enzymatically removed fraction, we prepared two affinity chromatography columns: one with Sepharose coupled to anti-MSA and another with anti-MMEC. Samples of the collagenase and hyaluronidase digest (table 4, step 1) were poured over each column, which was developed by the procedure described in the text. Of the material removed from the cell surface by this enzymatic digestion, 88% was bound to the anti-MMEC. In contrast, only 8% of the 3H-labeled material attached to the anti-MSA column (nonspecific binding). Specificity of Anti-MMFC

The data presented in table 5 reveal that the guinea pig anti-MMFC bound to MMFC; it did not detect MMEC or M-RBC. Furthermore, even if it was produced against cleared fat pad fibroblasts, it recognized those obtained from fetal tissues and, in the second stage of staining, yielded a fluorescence of equal intensity. DISCUSSION

Our present study clearly shows that some cell antigens are not altered during physiologic development of the gland in such a way that their reactivity toward specific antibodies is modified. The fact that these antigens can be removed by mild treatment with enzymes, a procedure that yields viable cells capable of regenerating these lost antigens in culture, suggests that they are either on the cell surface or very close to it. Thus anti-

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TABLE 3.-Regeneration of cell surface components on BALBlc MMEC as a function of time in suspension culture a Fluorescence

Suspension culture period, hr

Percent [3HIfucose incorporation d

Intensity" Percent" 0 0 0 0 6 0 0 7 18 61 1+ 35 24 72 2+ 51 48 93 4+ 83 72 90 4+ 100 a Cells were obtained from mid-pregnant BALB/c mice by method C; see text for details. " Percent = fluorescent-positive cells x 100. total cell seen by phase contrast Intensity was judged on a scale of 0 to 4+ (most intense). d 100% equals [3HIcpml ILg DNA at 72 hours. Values are averages of two closely agreeing determinations from different experiments. C

TABLE 4.-Surface components released during cell dissociation from mammary gland tissue labeled with ["Hjfucose a

Step

Control 1 2

3 4

Percent incorporation of ["HI fucose in

Treatment"

BSAd Collagenase + hyaluronidase Pronase DNase Suspension cultured, 2 days

Supernatant

Tissue residue

6 26

94 74

16 0

58 58

Fluorescence intensity"

0 0

0 4+

a For details of labeling procedure, see text. " See text for method C. " Fluorescence intensity of tissue residues or intact cells was determined as described in text and judged on a scale of 0 to 4+ (most intense). d Incubated for 2 hours with BSA (4%) at 37° C.

TABLE 5. -Specificity of anti-BALB Ie MMFC Cells MMFC MMFC MMEC MMEC M-RBC

Source Cleared fat pad Fetal tissue Pregnant gland Pregnant gland Blood

Dissociation method a A A

C C

Culture Monolayer, 14 daysC Monolayer, 14 days" Suspension, 2 days Monolayer, 10 daysC

Fluorescence intensity· 4+ 4+ 0

0 0

a Cells were dissociated by methods described in text. " Intensity was judged on a scale of 0 to 4+ (most intense). C Monolayer cultured cells were removed from the plates with a rubber policeman.

gens on the surface of MMEC are phenotypic traits whose formation remains unaltered by tissue culture and can serve as useful tools for cell identification. Early studies with tissue homogenate fractions demonstrated that many tissues possess organ-specific antigens (21-24). However, the methods used yielded an tigens that did not allow for the identification of the individual cell types in each organ. Recently, Schachner (25) produced antibodies to 3-methylcholanthrene-induced glioblastoma cells in mice and demonstrated that VOL. 57, NO. I,JULY 1976

IMMUNOLOGIC IDENTIFICATION OF MAMMARY CELL TYPES

only glial cells were reactive. Thus if one uses an enriched cell population for immunization, competition by other unwanted antigens is minimized. We have no evidence that the population of cells used to prepare anti-MMEC was homogeneous and not contaminated with other cell types. The procedure used to dissociate the mammary glands from pregnant mice enriches the resultant suspension with epithelial cells but does not insure a completely pure population. After these antibodies to the possibly mixed cell population were prepared, they were absorbed against M-RBC and MMFC to enhance the specificity of the preparation. Whether other cell types (e.g., myoepithelial cells or adipocytes) can react with the antibody is not certain. When pure preparations of these cells are obtained, we can, of course, absorb against them. In any case, the two antibodies (anti-MMEC and anti-MMFC) can each react with a different cell type to the exclusion of the other and thereby permit the identification of each cell type when grown in culture. The question arose whether the cell antigens, which are recognized by anti-MMEC, are indeed present on the cell when they are still part of the mammary tissue and not merely produced in culture. Cells removed from the tissue by gentle mechanical means could bind the anti-MMEC. This observation suggests that the antigen material is present on the cells in the tissue. Furthermore, when cells were grown in organ culture (in their own matrix) in the presence of [3H]fucose, labeled material was produced that could be bound to the antibody. Two observations indicate that fibroblasts obtained from fetal and mammary tissue possess similar antigen materials on their cell surfaces. The first was the finding that anti-MMFC does not differentiate between fibroblasts from fetal or mammary tissue. This does not prove that those cells are the same in all respects, but does demonstrate that they contain common cell antigens. The second indication comes from the experiments in which the binding ability of the anti-MMEC preparation for MMFC is removed by absorption with either fetal or mammary gland fibroblasts. REFERENCES (1) LASFARGUES EY: Cultivation and behavior in vitro of the normal mammary epithelium of the adult mouse. Anat Rec 127:117129, 1957 (2) - - - : Cultivation and behavior in vitro of the normal mammary epithelium of the adult mouse. II. Observations on the secretory activity. Exp Cell Res 13:553-562, 1957 (3) EBNER KE, HOOVER CR, HAGEMAN EC, et al: Cultivation and properties of bovine mammary cell cultures. Exp Cell Res

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23:373-385, 1961 (4) EBNER KE, HAGEMAN EC, LARSON BL: Functional biochemical changes in bovine mammary cell cultures. Exp Cell Res 25:555.1)70, 1961 (5) TWAROG jM, LARSON BL: Cellular identification of /3-lactoglobulin synthesis in bovine mammary cell cultures. Exp Cell Res 28:350-359, 1962 (6) RAO DR, HEGARTY HM, LARSON BL: Effect of cell density on lactose synthesis in bovine mammary cell cultures. j Dairy Sci 58: 159-163, 1975 (7) LARSON BL: Biosynthesis of milk. J Dairy Sci 52:737-747, 1969 (8) REIF AE, ALLEN jM: The AKR thymic antigen and its distribution in leukemias and nervous tissues. J Exp Med 120:413-433, 1964 (9) BOYSE EA, OLD LJ, STOCKERT E, et al: Genetic origin of tumor antigens. Cancer Res 28: 1280-1287, 1968 (10) OLD Lj, BOYSE EA: Antigens of tumors and leukemias induced by viruses. Fed Proc 24:1009-1017,1965 (11) KLEIN J, SHREFFLER DC: The H-2 model for the major histocompatibility systems. Transplant Rev 6:3-29, 1971 (12) BOYSE EA, STOCKERT E, OLD Lj: Properties of four antigens specified by "Tla" locus. Similarities and differences. In XXXVII International Convocation on Immunology, Buffalo, N.Y., 1968 (Rose NR, Milgrom F, eds.). Basel, New York, Karger, 1969, pp 353-357 (13) Kuo T, ROSAI J, TILLACK TW: Immunological studies of membrane glycoproteins isolated from human breast carcinomas. Int J Cancer 12:532-542, 1973 (14) DEOME KB, FAULKIN LJ, BERN HA, et al: Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads offemale C3H mice. Cancer Res 19:515-520, 1959 (15) WEIPJES GJ, PROP FJ: Improved method for preparation of single cell suspensions from mammary glands of adult virgin mouse. Exp Cell Res 61:451-454, 1970 (16) PITELKA DR, KERKOF PR, GAGNE HT, et al: Characteristics of cells dissociated from mouse mal)1mary gland. Exp Cell Res 57:4362, 1969 (17) GOLDMAN M: Staining methods. In Fluorescent Antibody Methods. New York, Academic Press, 1968, pp 153-172 (18) KISSANE J M, ROBINS E: The fluorimetric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system. j Bioi Chern 233:184-188, 1958 (19) WOFSY L, BURR BJ: The use of affinity chromatography for the specific purification of antibodies and antigens. J Immunol 103:380-382, 1969 (20) TAYLOR RB, DUFFIE WP, RAFF MC, et al: Redistribution and pinocytosis of lymphocyte surface immunoglobulin molecules induced by anti-immunoglobulin antibody. Nature 233:225229, 1971 (21) SULITZEANU D, YAGI Y, PRESSMAN D: Separation of an antigenic fraction from rat liver responsible for the formation of liver localizing antibodies. J Immunol 90:424-428, 1963 (22) BARNETT EV, DUMONDE DC, GLYNN LE: Induction ofautoimmunity to adrenal gland. Immunology 6:382-402, 1963 (23) MILGROM F, TUGGAC M, CAMPBELL WA, et al: Thermostable ethanol-insoluble antigens of brain. J Immunol 92:82-90, 1964 (24) MILGROM F, TUGGAC ZM, WITEBSKY E: Organ-specific antigens.of liver, testicle, and pituitary. j. Immunol 94:157-163, 1965 (25) SCHACHNER M: NS-I (Nervous system antigen-I), a glial-cellspecific antigenic component of the surface membrane. Proc Natl Acad Sci USA 71:1795-1799,1974

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FIGURES 1,2 .-Indirect fluorescence staining reactions, as detected with FIGaRYG as the amplifying layer and various absorbed rabbit y-globulin preparations. AI-DI represent the fluorescence staining reaction, and A2-D2 represent the same field under phase contrast microscopy. A) BALB/c MMEC (suspension cultured for 48 hr) plus normal rabbit y-globulin and FlGaR,u (nonspecific antisera control). B) Uncultured or freshly dissociated BALB/c MMEC plus rabbit anti-MMEC and FlGaR,u. C) BALB/c MMEC (48 hrs suspension cultured) plus rabbit antiMMEC and FlGaRyG • D) BALB/c MMFC (monolayer cultured for 14 days) plus rabbit anti-MMEC and FlGaRYG (control population of cells). Pictures were taken with Tri-X pan black and white film; exposure times were 1 min for fluorescence and 0.25 sec for phase contrast. x SOO

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