An immunocytotoxicity assay for neural cells in monolayer cultures

Prmted in Sweden Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827/79/060257-l 1%02.00/O

Experimental

Cell Research 120 (1979) 257-267

AN IMMUNOCYTOTOXICITY CELLS

ASSAY

IN MONOLAYER

FOR NEURAL

CULTURES

SILVIO VARON,’ ALICE M.-J. AU, ELEANORE and RUBEN ADLER

HEWITT

Department of Biology, School of Medicine, University of California, San Diego, La Jolla. CA 92093, USA

SUMMARY The immunological analysis of cell surface constituents which may characterize neuronal and glial populations, though still in its infancy, will greatly facilitate the investigation of several important problems in neurobiology. One critical component of such analyses is the way by which a given antiserum can be shown to be active on, and possibly selective for neurons and glial cells from normal neural tissues. This report describes the use of monolayer cultures of normal neural cells for recognition and quantitative titration of antisera directed against them. Sera were collected from rabbits immunized with chick embryo spinal cord cell suspensions, and found to be reactive to the same cells in the initial cell dissociate as well as in subsequent monolayer cultures of different in vitro ages. A monolayer assay procedure was developed, which (i) uses small numbers of cells and small volumes of immune reagents, with the possibility of further scaling down; (ii) applies equally to cultures using different substrata; (iii) permits differential counts of morphologically different cultured cells; (iv) allows to recognize cytological damage imposed by the immune serum in the presence, though not in the absence, of complement; and (v) quantitatively titrates the immune activity with lO- to 20-fold higher sensitivity than other titration procedures. While the study was not intended to investigate the possible specificities of the new antisera, it provided the unexpected observation that non-neuronal cells in these spinal cell cultures were considerably less sensitive than neurons to the complement-dependent action of the antisera.

Membrane surfaces are increasingly perceived as major sites for intercellular communications [14]. In the nervous system neuronal migration, guidance of growing axons, synaptic connectivity and glianeuron interactions, among other processes, are likely to occur via surface interactions and via recognition by one interacting element of surface features specific to the other. Immunochemistry provides a most sensitive approach to the recognition of subtle differences in biological molecules, and cell surface antigens are likely to reveal fine distinctions among different subclasses of neuronal and glial cells. In recent years, several laboratories have launched

attempts to generate antisera specifically directed to neural cell surface antigens. Used as immunogens were: cells from neural tumors grown in vivo [20,21]; clonal cell lines of neural origin [2, 3,8, 13, 15,23, 271; normal neural cell suspensions [ 16, 251 or their cultures [19, 261; membrane fractions from normal neural tissue [5, 6, 241or molecular constituents isolated from them [7,

181.

A critical component of such investigations is the way by which a given immune serum is verified to react with surface constituents of neural cells. In most cases, im1 To whom reprint requests should be addressed. Exp Cell Res 120 (1979)

Varon et al.

258

munoreactivity has been tested by: the loss sociated in fresh EBM,,, (0.5 ml/cord) by twenty passes through a Pasteur pipet. The cell suspension of reactivity incurred after adsorption on was filtered through a 20 pm nylon mesh. Final yields selected cell membranes [e.g. 8, 24, 271; the were6-8XIO6 cell/ml, or 3-4~ IO6cell/cord. complement-dependent abolition of dye-excluding properties in cell suspensions [8, zmmune Sera Spinal cord cells were centrifuged, resuspended in iso241; or inspection of histological sections tonic phosphate buffer (10 mM K,HPO, in I50 mM after exposure to antiserum coupled to NaCI), pH 7.0, refiltered on 20 pm nylon mesh, and reTwo ml aliquots, containing 12-15~ IO6cells, fluorescent dyes [8, 16, 24, 261 or horse- counted. were injected without adjuvant into the lateral ear vein radish peroxydase [22]. Monolayer cultures of two New Zealand female rabbits on days I, 7, 14, 36, and 70. The rabbits were bled from the central of normal neural cells offer opportunities artery of alternate ears (50 ml each time) at day Ofor both a visual inspection and a quantita- i.e. before injection (pre-immune serum)-and days 44, 58 and 77. The blood was allowed to clot in a tive assessment of the reactive cells, but 21, centrifuge tube for 2 h at room temperature. The clot their potential for immune analysis has not was loosened off the tube side and removed by 30 min at I700 g. Each serum (25-30 ml) was been tapped, thus far. The present report is centrifugation stored frozen (-20°C) in small aliquots. For use, a intended to provide an illustration of how serum aliquot was heated 30 min at 56°C for compleinactivation and stored at 4°C for up to 1 week. neural cell monolayers can be used to (i) ment The immune sera from successive bleedings of each recognize immune activity at a much rabbit are designated CESC- (for chick embryo spinal greater level of sensitivity than with some cord) 1A to ID and 2A to 2D, respectively. other techniques; (ii) determine quantitaMonolayer cultures tive titers of the immune activity; and (iii) Tissue culture plastic vessels were used, mainly 35 mm permit quantitative evaluations of differen- dishes (Falcon 3001) or 16 mm wells (MultiWell Plate, tial sensitivity to the same antiserum by dif- Falcon 3008). Polyornithine (PORN) coats were apolied bv dissolvina PORN (Nutritional Biochem.. ferent cell categories. This report is part of 5-20006 mol. wt) at-1 mglml in 150 mM borate buffer, an ongoing project aimed at utilizing anti- oH 8.4. diluting it IO-fold with H,O. distributing it to the vessels (1 and 0.5 ml to 35 mm’ dish and 1%mm bodies to neural cell surfaces as selective well, respectively), incubating overnight at room temligands for the physical separation of dif- perature, rinsing twice with H,O, and incubating another 30 min at 37°C with 1 ml culture medium prior to ferent neural cell classes [4]. cell seeding. Rat-tail collagen (0.5 mg protein/ml) was MATERIALS

AND METHODS

Media The following solutions and media were used: CMF, CaZ+, Mg*+-free saline [31]; EBM, Eagle’s Basal Medium (Earle’s salts) oreoared from nowdered EBM (Gibco) and supplemenied with glucose (to 6 g/l), glutamine (to 2 mM), penicillin (to 100 U/ml) and NaHCO, (to 0.2 g/l); HEBM, EBM further supplemented with bicarbonate to 2.2 all, for use in a 5% CO2 atmosphere; EBM, or EBM,,;EBM supplemented with 2 or 10% heat-inactivated fetal calf serum (FCS) (Gibco); culture medium, HEBM supplemented with 10% FCS.

Cell preparation The thoraco-lumbar portions of 8-day chick embryo spinal cords were collected in CMF, carefully freed from meningeal membranes, transferred to a 0.25% trypsin solution in CMF (0.5 ml/cord), incubated 30 min at 37”C, washed once with EBM,,, and disExp CdlRes lZO(1979)

aDDlied according to a Drocedure develooed in this laboratory [l] to generate a highly adhesive coat. The collagen (80 PI/dish or 20 PI/well) was spread over the plastic surface, polymerized by 30 min exposure to NH, vapors, incubated at 37°C in 5% CO,-air overnight with HEBM, and another 30 min whh culture medium prior to seeding. Spinal cord cell suspensions were diluted with culture medium to IO5cell/ml (about 70-fold), cell aliquots seeded (2 ml/dish. 0.5 ml/well) to achieve a seedine density of 250 cellimm2, and the cultures incubated a: 37°C in 5 % CO,-air. Differential cell counts were carried out, under phase contrast microscopy, over representative radial strips in dishes [31, 321 or diametral strips in wells. Additional details are given in Results.

RESULTS The spinal cord monolayer culture

Three main attributes were considered important for efficient use of monolayer cul-

Immunotoxicity tures in an immunocytotoxicity assay. One was the use of culture chambers small enough to require minimal amounts of the immune reagents (cells, antibodies, complement). The second attribute was adequate visibility under phase contrast microscopy for inspection and differential counting of the cultured cells. Lastly, culture conditions must provide for early attachment, survival and morphological expression of the several cell categories present in the test population, while also maintaining the cultured cells in a dispersed state so that differential cell counts could be conveniently carried out. The culture vessel. Preliminary work has led to the selection of the Falcon Multiwell plates (24 wells) as the culture container to be used in standard work. Each well, 16 mm in diameter, provides a 200 mm2 culture surface and uses one quarter of the cells required in a 35 mm dish. The culture can be carried with 500 ~1 medium, treated with 250 ~1 of antibody and complement mixtures, and analyzed with adequate visibility when filled with fluid to the top of the well (about 2.2 ml). The bottom of the wells can be easily coated with collagen or polycations, or used uncoated when the test cells would permit it. The prefabricated set of 24 wells makes for convenient set-up and handling of large number of cultures. However, investigative situations different from the one to be described in this report have to contend with two possible drawbacks. One is that complete filling of the well with fluid for phase contrast analysis greatly increases the hazards of contamination, and is better suited for terminal analysis of cultures to be discarded then for periodic analyses of ongoing ones. The other drawback would only be encountered if a great scarcity of the immune reagents were to require an even smaller scale of the assay system.

assay using neural cell cultures

259

With regard to scaling down, two other approaches have been explored for possible future applications. Glass rings, 1 cm inner diameter, can be cut from glass tubing and sealed to the bottom of plastic, 35 mm dishes (4 to a dish), each to provide a separate culture chamber. The sealing may be done with silicone grease [ 111,or by use of plastic solvent. In both cases the rings can be readily removed after treatment of the individual cultures so that phase contrast analysis can be applied to the open culture “patches”, immediately or after further culturing in a shared medium. Beside the inconvenience of having to assemble each 4ring dish, difficulties have been encountered in providing the bottoms of the individual chambers with uniformly spread collagen coats (though not with polycation coats). The “ring” chamber provides an 80 mm* culture surface, and requires 200 ~1 medium for carrying and 100 ,ul fluid for treating the cultures. An even greater scale reduction can be achieved by use of microtest plates (Costar or Falcon) containing 96 microwells, each having approx. 6 mm diameter and 30 mm2 surface and requiring 100 ,ul and 50 ~1 fluid for carrying and, respectively, treating the cultures. As with the 16 mm wells, phase contrast visibility can be obtained by filling completely the microwells with fluid. However, as with the ring chambers, collagen coating has not been satisfactorily achieved, thus far. The culture substratum. Highly adhesive substrata offer, in principle, several advantages. Attachment of the seeded cells occurs within minutes, preventing cell reaggregation prior to attachment [ 11. The attached cells have limited mobility and, thus, remain dispersed and countable for a longer period of time in culture, while neuritic outgrowth is unrestrained or even promoted [l, 10, 301. Lastly, proliferation of non-

260

Varon et al.

neuronal elements may be retarded or altogether blocked [ 1,301. Associated with such advantages, however, may be two disadvantages: (i) the assumption by nonneuronal cells of typically stretched shapes is also delayed, which may hamper cell recognition and differential cell counting; and (ii) dead cells and their debris also attach and persist, causing the effects of an immunocytotoxic treatment to be less readily quantifiable. Two highly adhesive substrata were examined for use with chick embryo spinal cord cells, although future work with other cell populations may encourage the use of other substrata. Polyornithine (PORN) has been profitably used for cells from chick embryo dorsal root ganglia [lo] and ciliary ganglia [9, 331, and was applied in the present study at 0.1 mg/ml (see Methods). Collagen has been a favorite substratum for neural cell cultures from both peripheral and central neural sources [28], although important differences in cell behavior can derive from minimal differences in the coating technique [29]. In the present work we used the highly adhesive, A 30-O coating technique [I]. Decreasing adhesiveness may be imposed by interposing increasing numbers of washes between the ammoniation and overnight incubation steps [I].

neuronal “flat” cells and a still considerable number of rounded elements not readily attributable to either category. At 3 days, both neuronal and non-neuronal cells are well developed, while still sufficiently dispersed for accurate differential counts. In older cultures, flat cell numbers increase rapidly to eventual confluence, neurons tend to cluster and neurites fasciculate into interconnecting fiber bundles, so that differential counting is increasingly difficult. As will be described further on, l-day old cultures on collagen were chosen for routine analysis of CESC antisera, while similar but older cultures were used to evaluate differential immune effects on neuronal and non-neuronal cell elements. Preliminary examination immune serum

of

CESC-2D, one of the rabbit antisera produced against chick embryo spinal cord cells (see Methods), was used for preliminary investigations of its presumptive immune activity. Table 1 offers a comparison of results obtained by three different approaches. Immunojluorescence. Spinal cord cells were seeded on PORN-coated glass coverslips (lying on the bottom of 35 mm dishes). One hour later, the cultures were washed once with EBM buffered with 3.34 mg/ml Cell behavior in the spinal cord monoN-2layers. The present study was concerned Hepes (N,-hydroxyethylpiperazine, strictly with short-term analyses suitable ethanesulfonic acid) pH 7.5 and supplefor immunocytotoxicity tests. Spinal cord mented with 2% FCS (Hepes-EBM,). The cells were seeded on either collagen or coverslips were transferred to a slide, then PORN substrata and the cultures examined covered with 200 ~1 of antiserum at the after l-5 days in vitro before and after treat- selected final dilutions in Hepes-EBM,, inment with an effective mixture of antiserum cubated 60 min at room temperature, and complement (see further on). With both washed 3 times with Hepes-EBM,, fixed substrata, 1 day old untreated cultures dis- with acetone, and treated 30 min at 37°C play reasonably dispersed neuronal ele- with fluorescein isothiocyanate-conjugated ments with already some neuritic develop- goat antiserum against rabbit IgG (Miles ment, accompanied by relatively few non- Laboratories) diluted 1: 20 in Hepes-EBM,. Exp CeNRes IZO(1979)

Immunotoxicity

assay using neural cell cultures

Table 1. Comparative

evaluation, by different methods, of the activity serum (CESC-2D) produced against chick embryo spinal cord cells

261

of an immune

Dilution factors Evaluation methods (a) Indirect-immunothrorescence (b) Trypan blue exclusion (% of dye-positive cells) (c) Monolayer assay (% damaged cells) CESC-2D+complement CESC-2D-complement Complement alone (1 : 20)

800

I 600

0

-

-

100

35

10

=O

loo 5

100 0

100 0

100 0

25

50

100

400

+++

+

0

100

100

None

All methods used as test cells the population derived from chick embryo spinal cord (a) after 1 h attachment to a polyomithine-coated coverslip; (b) in the original suspension, or (c) 2 days in culture on a collagen substratum. For details, see text.

After 3 more washes (5 min each), the covet-slips were examined under a fluorescence microscope, using a Leitz BG,, excitation and Ksl,, suppression filters. With minor modifications, this was the procedure used in this laboratory for intracellular antigens [ 121.No attempts were made to quantitate the number or the intensity of the fluorescent cells. Fluorescent cells were still detectable at 50-fold dilutions of CESC2D, but no longer so at loo-fold dilutions. No fluorescence was observed when preimmune serum (from the same rabbit) was used at IO-fold dilution. While this approach was adequate to demonstrate affinity of the CESC immune serum for spinal cord cells, it clearly showed considerable limits in sensitivity and practicality for a quantitative immune assay, at least in its present form. Trypan blue exclusion. Freshly prepared spinal cord cell dissociates were diluted to 3 x lo6 cell/ml (about two-fold) with HepesEBM*. Two-fold serial dilutions of CESC2D serum were prepared in the same medium. Fifty ~1 aliquots of cell suspension were mixed with 50 ~1 of diluted antiserum and 50 ~1 of rabbit complement (Gibco, diluted 1 : 20 in the same medium), incubated 45 min at 37°C in air, and transferred

to an ice bath. Hundred ~1 of a 0.4 % saline solution of Trypan Blue (Gibco) were added to two tubes at a time. After mixing, the cell samples were counted in a hemocytometer for total and Trypan blue-positive cells, and the results corrected for the number of positive cells found in control tubes (untreated, or treated with antiserum alone or complement alone). All cells took up the dye in the presence of complement, when treated with antiserum diluted up to 100x, while 1600x diluted antiserum did not cause appearance of dye-positive cells in excess of control levels. Intermediate dilutions suggested a 50% effect at about 200~ dilution. Thus, by this traditional assay, CESC-2D displayed considerable immune activity in a range not dissimilar from that of many other antisera to neural cell surface antigens [e.g. 16, 24; however, see 81. Monolayer assay. Spinal cord cells were seeded into collagen-coated 35 mm dishes, cultured for 2 days, washed once with Hepes-EBM,, and then presented with 1 ml of this medium containing serially diluted CESC-2D antiserum plus 20-fold diluted rabbit complement (or antiserum alone, or complement alone as controls). After 60 min incubation at 37°C in air, several radial Exp CdRes 120(1979)

262

Varon et al.

Fig. I. Phase contrast photomicrographs of spinal cord cell cultures on collagen, 24 h in vitro. (A) Control cultures, showing a large majority of healthy neurons (note one non-neuronal cell in the upmost right frame). Two “damaged” cells are indicated by the open ar-

rows; (B) cultures treated with CESC antiserum plus complement, showing mostly “damaged” cells. Two “undamaged” survivors are indicated by the solid arrows. x300.

Immunotoxicity assay using neural cell cultures strips were counted under phase contrast microscopy for “undamaged” cells, and the results recalculated as percentages of “damaged’ cells relative to the viable cells present in control cultures. Fig. 1 illustrates the appearance of viable and damaged cells in control (top) and treated (bottom) cultures. Viable or undamaged cells are plump, phase-bright elements frequently displaying processes (neurons) or-in rare cases within early cultures-elements with the elongated flat morphology typical of nonneuronal cells. After immune treatment, the cultures exhibited fewer cells, most of which had lost their plumpness, had a crinated or partially fragmented appearance or had converted to flat, circular “ghosts”. Counting of cells that retained the “undamaged” appearance was a relatively easy task, with only a modest number of elements requiring a subjective judgement. As shown further on, this afforded a practical and reproducible assessment of the immunocytotoxic treatment. Further veritication of the cytotoxic effectiveness of antiserum treatment was obtained by replacing the treatment media (after the 60 min exposure) with fresh culture medium, and inspecting the cultures in subsequent days. No living cells were detected over a week period after the immune treatment even when attempted on 1 h old cultures. In contrast, cultures treated with antiserum alone, or complement alone, exhibited essentially the same neuronal and non-neuronal behaviors as those of altogether untreated cultures. At the dilutions tested, up to 1600x (table l), the CESC-2D serum damaged the entire cell population in the presence of complement, but few or no cells in its absence. No additional damaged cells were detected on treatment with complement

263

alone. Thus, the monolayer assay system effectively demonstrated the immune reactivity of the CESC serum and, in addition, proved to be considerably more sensitive than the dye exclusion test applied to the original cell suspension. The monolayer assay was used to examine complement fractions from several sources, either alone to measure their direct toxicity, or in combination with 1600-fold diluted CESC-2D to measure their immunocytolytic effectiveness. Rabbit complement (Gibco) was not toxic, was fully effective at 1 : 40 dilution, and was adopted for future routine use. Rabbit complement (PelFreeze) and guinea pig complement (PelFreeze) were also suitable, at 1 : 30 dilutions. Immunotitration on monolayer cultures The following procedure was developed for routine analyses of immune reagents. Spinal cord cells (50000 in 0.5 ml culture medium) are seeded into collagen-coated 16 mm wells, and cultured for 24 h. The cultures are washed once with EBM*, and treated 45 min at 37°C with 250 ~1 of EBM, containing the desired amount of antibody as well as rabbit complement (I : 40 final dilution). After another wash with 500 ~1 EBM2, the wells are filled with 2% glutaraldehyde in serum-free EBM, and undamaged cells are counted under phase contrast microscopy. Use of glutaraldehyde does not affect cell morphology and conveniently extends for l-2 days the availability of the treated cultures for cell counting. First, the activity range of a given material is determined with IO-fold serial dilutions. Next, a second series is run at smaller dilution steps within the active range, using at least duplicate wells for each dilution and control wells with complement only or the immune material only (at full activity). The results are E-r/, CrllRr~ I20 (1979)

264

Varon et al.

7000

t 6000

t 5000 t

4000 3000

50%-

I 2ooot

2 4

16

6 10

20

24

AS C'

t

Fig. 2. Abscissa:

dilution factors (~10~~); ordinate: undamaged cells/well. Monolayer titrations of CESC-2D antiserum. Each solid curve derives from an independent experiment. Broken line represents their average. Large circle denotes the 50% effect, and the corresponding arrow indicates the titer (I 1000 NJ/ml) assigned to the antiserum. Right-side bars, undamaged cells in wells treated with antiserum only (AS), or with complement only CC’).

plotted, against the dilution factors used, as total number of undamaged cells or as % of viable cells in the controls. The dilution required for 50% cell damage is chosen to represent the immune activity of the test material. Thus, one immune unit (IU) is delined as the antibody activity present in 1 ml of assay medium yielding 50% cell damage in the monolayer assay system. The original material, therefore, contains a number of III/ml equal to the 50% effective dilution factor. Fig. 2 illustrates the titration system for the CESC-2D antiserum. The three solid curves were derived from three independent titrations, using different cell batches and different dilution sets. The broken curve represents the average of the three individual assays. The “monolayer” titer of CESC-2D was 11000 III/ml, with about 10% variations on either side among the individual runs. ExpCdRes

120(1979)

Fig. 3 compares titration curves obtained for CESC-2D with monolayer cultures on collagen (solid lines) and PORN (broken lines), after 1 day (closed symbols) and 3 days (open symbols) of incubation. Both substrata yielded similar results, demonstrating the possibility of using either one for monolayer assays of immunocytotoxicity. The titer was also not significantly altered by the use of cultures of different in vitro age, indicating that the 20-fold higher titer yielded by the monolayer assay as compared with the dye-exclusion assay (table 1) was not likely to be due to an increased availability with time of reactive antigens on the surface of cultured cells. In experiments using 1 to 3 day-old cultures, it was noted that recognizable nonneuronal elements often resisted treatments with CESC-2D at dilution factors higher than 4000x. Since the numbers of these non-neuronal “flat cells” (thinly spread phase-light, irregularly contoured cells) is very modest in the early cultures, cultures were carried for 3, 4 or 5 days, treated with antiserum and complement, and analyzed separately for neuronal and flat cells. Fig. 4 shows that titration curves on neurons only

Fig. 3. Abscissa:

dilution factors (X 10m3);ordinute: % undamaged cells relative to controls. Monolayer titrations of CESC-2D antiserum on collagen (solid lines) or polyornithine (broken lines) substrata, at either I day (close symbols) or 3 day (open symbols) in vitro.

Immunotoxicity assay using neural cell cultures

a

12

10

24

Fig. 4. Abscissa: dilution factors (X lO-3); ordinates: undamaged cells/well. Comparative effects of CESC-2D antiserum on neurons (left side) and nonneuronal “flat” cells (right side) in monolayer cultures at 3 (O-O), 4 (O-O) or 5 (X-X) days in vitro.

44

1

4

14

8

12

(left side) were essentially the same for all three culture ages, yielding titers between 10000 and 12000 IU/ml antiserum. The non-neuronal data (right side) revealed a very different pattern. Flat cells increased about &fold from 3rd to 5th day, as expected from their proliferative capability. At each age, the number of viable cells was not at all affected by exposure to CESC-2D antiserum at dilution factors of 4000x or greater. At dilution factors lower than 2000x however, flat cells remained sensitive to the complement-dependent action of the antiserum, and all of them were damaged at 500~ dilutions. These results offer two noteworthy, though only preliminary suggestions: (i) the monolayer assay permits the recognition of immunoreactive differences between the surfaces of different cell classes; and (ii) the antigenicity of the non-neuronal cells was not noticeably different in the later progeny, as compared with the earliest recognizable cell elements. Finally, the eight CESC antisera obtained by immunization of two rabbits with spinal cord cells were titrated for total cell immunocytotoxicity in the routine 24 h old monolayer system. The results are presented in table 2. All eight sera had very low intrinsic toxicity (data not shown), but exhibited high complement-dependent cytotoxicity, with titers ranging from 9000 to

18

265

24

16000 IU/ml. No correlation pattern was apparent with regard to the immunization schedule, except possibly for the low titers of the two sera obtained with the 3rd bleeding (CESC-1C and CESC-ZC), which was separated from the last preceding immunogen injection by three weeks instead of the customary one. DISCUSSION The work reported here demonstrates the possibility of using monolayer cultures of normal neural cells for both the detection and the titration of immunological activity directed to such cultured cells. Comparison of antiserum titers derived from the new

Table 2. Monolayer immune titers of the several sera obtained from two rabbits (CESC-I and CESC-2) immunized with chick embryo spinal cord cells Titers (W/ml)

1st bleed (A) 2nd bleed (B) 3rd bleed (C) 4th bleed (D)

CESC-I

CESC-2

II ooo

16 000

16 000 9 600

15 600

16000

II 400

9000

For immunization and bleeding schedules, see Materials and Methods. Exp Cell Res I20 (1979)

266

Varon et al.

monolayer assay with those obtained by more traditional dye exclusion tests indicates a IO-20 times greater sensitivity with the culture system. The monolayer assay can be used directly with immune sera, or with immune globulin fractions derived from them-allowing for quantitative determinations of the immune activity recovered in the course of purification procedures [4]. The assay appears to work equally well with cultures using highly adhesive collagen or polyornithine as substrata, providing options for the different culture systems to which it may be extended in the future. Conditions have been worked out to make the monolayer assay both practically convenient and economical in terms of its requirements for test cells and immune reagents. Chick embryo spinal cord cells have been found to be good immunogens in the rabbit. All eight sera obtained were immunologically active, with high monolayer immune titers (9-16000 IU/ml). It is important to stress that the immune activity examined by the monolayer assay is that directed to surface antigens of the test cells. A more detailed examination of one antiserum showed it to be reactive to freshly dissociated cells (dye exclusion test) such as those previously used as immunogens, to cells cultured for 1 h (immunofluorescent test, monolayer test), and to cells cultured for several days (monolayers of different in vitro ages). Thus, the surface antigens to which the rabbit had reacted (i) are resistant to trypsin; and (ii) persist in culture for several days, possibly even with no quantitative changes. It was not the purpose of this study to examine the nature or the cell specificity of the surface antigens recognized by the new antisera, and no information was sought as to whether the antisera obtained comprise Exp Cd Res 120(19791

antineural or merely anti-chick antibodies. Nevertheless, it was unexpectedly observed with at least one antiserum that its activity against cultured non-neurons was lost at dilutions that were still effective on neuronal elements. Future studies may clarify whether the same antigens were available for immune recognition at higher levels on neuronal than on non-neuronal surfaces, or whether qualitatively different antigens of the two cell classes were recognized by different antibodies in the same serum. In either case, the observation indicates the potential ability of monolayer assays to distinguish among cells with different immunoreactivities. The use of monolayer neural cell cultures as immunocytotoxicity assay systems provide opportunities in several directions. Among those to be pursued in this laboratory are: (i) Characterization of the antisera newly obtained against chick embryo spinal cord cells, after adsorption on appropriate tissues of the activity which is not specifically directed to neural cells. In addition, the availability in this laboratory of several purified neuronal and non-neuronal cell cultures [I, 12, 17, 31, 321 could make it possible to segregate immune activities directed to neurons and non-neurons of the same tissue. (ii) Establishment of a battery of test cultures (using cells derived from a variety of peripheral and central neural tissues from chick and rodent), to screen a number of antisera raised in this and other laboratories against normal or tumora! neural cell surface antigens. Such a screening would provide a profile of the activity of each serum on normal neural cells and, conversely, open the way to antigenic mapping of neural cell surfaces in different cell classes. (iii) Application of immune reagents with

Immunotoxicity

recognized specificity for neuronal or glial cell surfaces to the separation of viable neural cell populations by immunoaffinity methods [4]. This work was supported by USPHS grant NB-12893 from the National Institute of Neurological and Communicative Disorders and Stroke.

REFERENCES 1. Adler, R, Manthorpe, M C & Varon, S, Dev biol (1979). In press. 2. Akeson, R & Herschman, H R. Nature 249 (1974) 620. 3. Akeson. A & Seeger, R C. .I immunol II8 (1977) 199.5. 4. Au, A M-J & Varon, S, Exp cell res 120 (1979) 269. 5 . Bock, E, Cell tissue and organ cultures in neurobiology (ed S Fedoroff & L Hertz) p.. 407. Academic Press, New York (1977). 6. Bornstein, MB & Raine, CA, Lab invest 23 (1970) 536. 7. Fry, J M, Lisak, R P, Manning, M C & Silberberg, D H, J immunol meth I1 (1976) 185. 8. Fields, K L, Gosling, C, Megson, M & Stern, P L, Proc natl acad sci US 72 (1975) 1296. 9. Helfand, S L, Smith, G A & Wessells, N K, Dev biol 50 (1976) 541. 10. Letourneau, P C, Dev bio144 (1975) 77. II. McCarthy, K D & Partlow, L M, Brain res I14 (1976) 391. 12. Manthorpe, M C, Adler, R & Varon, S. Submitted for publication. 13. Martin, S E & Martin, W J, Cancer res 35 (1975) 2609. 14. Moscona, A A (ed), The cell surface in development, Wiley, New York (1974).

assay using neural

cell cultures

267

IS. Pfeiffer. S E. Herschman. H R. Liahtbodv. J E.

Sato, G & Levine, L, J cell physiol 78 (1971) 145. 16. Poduslo, S E, McFarland, H F & McKhann. G M, Science 197(1977) 270. 17. Popiela, H, Manthorpe, M C, Adler, R & Varon, S, Trans Am sot neurochem 9 (I 978) 49. 18. Rapport, M M & Karpiak, S E, Trans Am sot neurochem 8 (I 977) 200. 19. Rutishauser, U. Brackenbury. R, Thiery, J P & Edelman, G M, Molecular basis of cell-cell interaction (ed R A Lerner & D Bergsma) p. 305. Liss, New York (1977). 20. Schachner. M, Proc natl acad sci US 71 (1974) 1795. 21. Schachner. M & Camow. T B, Brain res 88 (1975) 394. 22. Schachner, M, Rubert, M Z & Carnow. T B, Brain res bull I (1976) 367. 23. Schachner, M & Wortham, K A. Brain res 99 (1975) 201. 24. Schachner, M. Wortham, K A, Carter, L D & Chaffee, J F, Dev bio144 (1975) 313. 25. Schachner. M. Wortham. K A. Rubere. M Z. Dorfman, S & Campbell, G LeM, Brainres 127 (1977) 87. 26. Seeds, NW, Proc natl acad sci US 72 (1975) 4110. 27. Stallcup, W B & Cohn, M, Exp cell res 98 (1976) 285. 28. Varon, S, Exp neurol48 (1975) 93. 29. Varon, S, Cell tissue and organ cultures in neurobiology (ed S Fedoroff & L Hertz) p. 237. Academic Press, New York (1977). 30. Varon, S, Neurochem res. In press. 31. Varon, S & Raiborn, C, J neurocytol I (1972) 21 I. 32. Varon, S. Raiborn, C & Tyszka, E, Brain res 54 (1973) 51. 33. Varon, S, Manthorpe, M C & Adler, R, Brain res (1979). In press. Received September 15, 1978 Accepted November 22, 1978

Exp CellRc.s I20 (1979)