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Measurement of cell numbers by means of the endogenous enzyme hexosaminidase. Applications to detection of lymphokines and cell surface antigens
Journal oflmmunologicalMethods, 67 (1984) 379-388
379
Elsevier JIM03011
Measurement of Cell Numbers by Means of the Endogenous Enzyme Hexosaminidase. Applications to Detection of Lymphokines and Cell Surface Antigens Ulf Landegren Department of lmmunology, Biomedical Center, Uppsala University, Box 582, S-751 23 Uppsala, Sweden
(Received 27 June 1983, accepted 1 December 1983)
By using a chromogenic substrate for an ubiquitous lysosomal enzyme, hexosaminidase to estimate cell numbers, a sensitive and simple procedure has been developed in which microtiter reaction wells are directly scanned in a spectrophotometer. This method has been adapted to several cell biological assays. Quantitation of the biological activities of T cell growth factor and interferon can be performed on large numbers of samples. Adhesion of dispersed solid tissue cells to fibronectin coated substrates may be quantitated with little expenditure of reagents. By use of a panning procedure in microtiter plates a sensitive and very simple assay for the binding of monoclonal antibodies to cell surface antigens has been developed. Key words: cell quantitation - growth factor assay - cell surface antigens - cell adhesion - endogenous enzyme assay
Introduction The enzyme N-acetyl-fl-D-hexosaminidase (2-acetamido-2-deoxy-fl-D-glucoside acetamidodeoxuglucohydrolase, EC 3.2.1.30) is a lysosomal enzyme thought to be involved in the d e g r a d a t i o n of glycosylated cellular constituents. It has been d e m o n strated in m a n y tissues a n d in different phyla. A p - n i t r o p h e n o l derivatized substrate can be employed for the spectrophotometric q u a n t i t a t i o n of the activity of this enzyme (Banerjee a n d Basu, 1975). Its widespread occurrence a n d its catalytic activity make this enzyme a n ideal indicator for analysis of cell n u m b e r s . It was first used in this way b y K o p o n e n et al. (1982) to score the sensitivity of a series of t h y m o m a cell lines to the growth i n h i b i t o r y action
Abbreviations." hexosaminidase,N-acetyl-fl-D-hexosaminidase;PBS, phosphate-buffered saline; TCGF, T cell growth factor.
0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.
380 of cyclosporin A. Measurements of this enzyme in cell populations heterogeneous with regard to differentiation stage and lineage need to be analyzed with the caveat that different cells may contribute variably to the overall enzyme reaction. With this limitation in mind, a number of assays, where an effect upon cell numbers is sought, may be accommodated to measurement of this enzyme. I describe here its use for the measurement of cell growth, protection from lysis and binding to substrates via cell surface molecules. Each of these assays is analysed by a number of techniques, commonly involving the use of isotopes. The methods described here allow less manipulation of the samples with good exactitude in determination. In addition, the methods are not dependent upon expensive or unstable reagents.
Materials and Methods
Reagents The substrate for hexosaminidase, p-nitrophenol-N-acetyl-fl-D-glucosaminide was obtained from Sigma (St. Louis, MO). Purified human T cell growth factor (TCGF) was generously supplied by Dr. Richard Robb, Glenolden Laboratories, Glenolden, PA. Human a-interferon was provided by Dr. Gunnar Alm, Departments of Immunology and Medical Virology, BMC, Uppsala. Human fibronectin was a gift by Dr. Kristofer Rubin, Department of Immunology, BMC, Uppsala. F(ab)2 fragments of goat anti-mouse IgG was from Cappel, Cochranville, PA. The OKT 9 monoclonal antibody used had specificity for transferrin receptor (Sutherland et al., 1981) and was obtained from Ortho Pharmaceutical, Raritan, NJ. The 3Cll hybridoma was established in this laboratory by Lena Sporrong. It produces antibodies with affinity for human T cells. Cell culture All cultures were performed in RPMI 1640 with 5% fetal calf serum and supplemented with 2 mM L-glutamine, 50 #M 2-mercaptoethanol, 50 I U / m l penicillin and 50/xg/ml streptomycin. The cell lines investigated were Jurcat, a human T leukemia line (Gillis and Watson, 1980), Raft, a Burkitt lymphoma line, CTLL-2, a T cell growth factor (TCGF) dependent mouse line (Baker et al., 1979) and WISH, a human amniotic cell line. Blood was obtained from volunteers. The cells were separated over Ficoll-Hypaque gradients and the peripheral blood lymphocytes at the interface were employed. Hepatocytes were obtained from rat liver by collagenase perfusion (Rubin et al., 1979). The hexosaminidase reaction The substrate for the enzyme hexosaminidase, p-nitrophenol-N-acetyl-fl-D-glucoseaminide, was dissolved at 7.5 mM in 0.1 M citrate buffer, pH 5. The solution was then mixed with an equal volume of 0.5% Triton X-100 in water, aliquoted and
381
stored at - 1 8 ° C . The substrate solution was added in volumes of 60/~l to cells in flat bottom microtiter wells. The plates were then incubated at 37°C in 100% humidity. After a suitable interval, the color reaction was developed and enzyme activity blocked by addition of 50 mM glycine buffer, p H 10.4, containing 5 mM EDTA, 90/tl per well. Absorbances were measured in a Titertek ® multiskan at 405 nm.
Proliferation assay Cells were grown in flat bottom microtiter plates (Nunc, Denmark). For the thymidine incorporation assay, 2 ~tCi [methyl-3H]thymidine was added per well for the last 2 h of culture. The cells were then harvested on glassfiber paper and processed for scintillation counting. For the enzyme assay, culture was terminated by spinning the plates at 2000 rpm for 5 min. The supernatant was removed by rapidly inverting the plate. Phosphate-buffered saline (PBS) was added and the plates were centrifuged. After flicking the plates again, substrate was added and the enzyme reaction was allowed to proceed for 4 h.
Interferon assay The assay was performed according to R6nnblom and Alm (1982). Briefly, WISH cells were grown in monolayers and exposed to dilutions of interferon. Virus was then added to the wells and culture continued. At the end of incubation, the cells were fixed and stained with crystal violet to determine percentage protection by examination with an inverted microscope. Alternatively, the substrate for hexosaminidase was added and the color reaction measured after 45 min incubation.
Substrate adhesion of hepatocytes Adhesion of hepatocytes was measured by incubating cells for 1 h at 37°C in microtiter wells that had been coated with fibronectin or bovine serum albumin for 1 h at 37°C (Rubin et al., 1979). The wells were washed by immersing the plates in buffer and flicking off unbound cells. After 2 washes, substrate was added for quantitation of cells remaining in the wells.
Detection of antibody binding to cell surface antigens Flat bottom microtiter plates were coated with F(ab)2 fragments of affinity purified goat anti-mouse IgG, 100/~g/ml in 0.05 M Tris buffer, pH 9.5, for 1 h at 37°C. Forty/~l were added per well. The plates were washed 3 times with PBS before the addition 30/~l/well of monoclonal antibodies in medium. Usually a concentration of 1/~g/ml or a 1/10 dilution of hybridoma supernatant was used. After 1½ h at 37°C the plates were washed again. Cells to be tested for adherence to monoclonal antibody coated plates were added in 100/xl, 2 × 105 per well. Hates were centrifuged at 500 rpm for 2 rnin and then incubated for 1½ h at 4°C. At the end of incubation, the plates were washed by immersing in cold PBS and flicking off buffer. Usually no non-specifically adherent cells remained after 3 cycles of washes. This was monitored in an inverted microscope. Quantitation of cells was performed by incubating with substrate and determining absorbancy.
382
Results
The absorbancy of the product of the hexosaminidase reaction is directly proportional to cell numbers and the time allowed for the reaction For analysis of hexosaminidase content, cells were added to microtiter plates in serial dilutions. The plates were centrifuged and the supernatant removed. Substrate was added and the absorbancy of the colored product of the enzyme reaction was determined. In Fig. 1 the 3 lymphocyte lines, CTLL-2, Jurcat and Raji are seen to express similar amounts of enzyme activity. Peripheral blood lymphocytes contain half as much and hepatocytes 5 times as much activity on a per cell basis. CTLL-2 cells were titered in 4 replicate plates and were then exposed to the substrate for variable periods of time. In Fig. 2 the absorbancy is seen to be directly proportional to the number of cells in the well. Furthermore, absorbancy is linearly dependent upon the time allowed for the reaction. When the time for the enzyme reaction was increased 4-fold, one fourth the number of cells are sufficient to give similar absorbancy. One of the plates was stored at - 18°C for 2 days before the 4 h enzyme reaction was carried out. This did not measureably affect enzyme activity. When the results shown in Figs. 1 and 2 are approximated to straight lines, the mean slope is 0.988 + 0.029 SD with a mean correlation coefficient 0.998 + 0.001 SD, indicating direct proportionality between cell numbers and absorbancy. In the calculations, absorbancies between 0.050 and 2.0 have been employed. This allows determination of cell numbers over a 40-fold range for a given time of exposure to the substrate.
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Fig. 1. Hepatocytes (O), CTLL-2 cells (13), Jurcat cells (zx), Raji cells (O), and peripheral blood lymphocytes ( * ) were diluted 2-fold in triplicate in microtiter wells. After removing the supernatant, the substrate for hexosaminidase was added and the plates incubated for 2 h at 37°C. The color reaction was developed and the absorbance determined after blanking the machine on wells that did not contain cells. Points are + SD.
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CELLSI WEll Fig. 2. CTLL-2 cells were diluted 2-fold in quadruplicate in 4 replica microtiter plates. The enzyme was allowed to work on the substrate for 1, 4 or 16 b before the reaction was blocked. A fourth plate was stored at - 1 8 ° C for 2 days before being incubated with substrate for 4 h. The absorbancies of the individual wells are represented by circles. The results have been fitted to straight lines and are shown with confidence intervals ( P < 0.05). Frozen samples are represented by filled circles and are approximated with the dashed line.
Measurement of T cell growth factor activity by quantitation of cells When TCGF is estimated by its ability to induce DNA synthesis in TCGF dependent cells, optimal results are obtained if the thymidine pulse is given 24 h after initiating the culture. At this time any incorporation of tritiated thymidine is 1.5
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Fig. 3. Four × 104 CTLL-2 cells/ml in 2-fold dilutions of T C G F from 2.5 U / m l were cultured for 24 h before being assayed for incorporation of tritiated thymidine during 2 h (El), or for 48 h before being washed once and then exposed to the substrate for hexosaminidase for 4 h. Absorbancy was determined after blanking the machine on wells that did not receive T C G F (O). The means and SDs of triplicate determinations are shown.
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Fig. 4. WISH cells treated with the indicated concentrations of interferon were challenged with virus at dilutions of 1/400 (t3), 1/1600 (~), 1/6400 (zx) or with no virus (O). Replicate microtiter plates were then (A) assayed for hexosaminidase after 45 min incubation with substrate or (B) fixed and stained and visually scored for protection of individual wells in an inverted microscope. Means of triplicates are given _+SD.
dependent upon T C G F present in the assay while little T C G F will have been consumed. If instead of measuring D N A synthesis, the T C G F dependent survival and expansion of cells is investigated via the hexosaminidase assay, this is optimally done after 48 h of culture. In Fig. 3 results obtained by thymidine incorporation and by the hexosaminidase assay are compared. The sensitivity is similar, but the enzyme assay is more precise and less laborious.
Quantitation of interferon by monitoring hexosaminidase actioity Interferons are estimated by observing their ability to protect virally challenged cells from lysis. This is commonly scored by micro- or macroscopically inspecting the stained cells for protection at the end of the assay. In Fig. 4 results by this method are compared with those by the alternative strategy of quantitating hexosaminidase in cells remaining at the end of culture. Interferon was titered against different doses of virus. While both measurements demonstrate the effect of interferon, the hexosaminidase measurements gives greater variability. In addition a weak cytopathic effect observed at 12.5 I U interferon/ml could not be demonstrated spectrophotometrically. The advantage of the enzyme technique, however, lies in its objectivity and rapidity.
Determination of the ability of hepatocytes to bind to immobilized fibronectin Solid tissue cells carry surface molecules enabling them to adhere to substrates coated with extraceUular matrix macromolecules such as collagen or fibronectin (Applin and Hughes, 1982). The hexosaminidase reaction allows convenient quantitation of adhering cells. In Fig. 5 results are given of an experiment where 2 concentrations of hepatocytes were allowed to settle in wells previously coated with
385 1.5
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Fig. 5. Adhesion of hepatocytes to wells coated with the indicated concentrations of fibronectin (O, e) or bovine serum albumin (O, I). Three× 105 (O, D) or 1 × 105 (O, II) cells were added per well. After the plates had been washed twice, the substrate for hexosaminidase was added for 1½ h. The symbols represent mear.___SD of duplicate determinations.
fibronectin or bovine serum albumin. Concentrations of fibronectin from 10 to 250 /~g/ml permitted stable binding to the microtiter wells. These figures are in accordance with published data (Rubin et al., 1979). Demonstration of cells binding to monoclonal antibodies immobilized in microtiter wells Several techniques are used for analysis of monoclonal antibodies binding to cell surface antigens. A method which combines simplicity and sensitivity with the possibility of avoiding fixing the cells would be valuable for developing hybridomas as well as for investigation of cell surface antigens. For this reason the panning method of Wysocki and Sato (1978) was adapted to microtiter plates. If purified anti-mouse immunoglobulin antibodies are allowed to bind to the wells, cell specific monoclonal antibodies, when bound by the anti-mouse immunoglobulin, will effectively immobilize cells added to the wells, provided the cell surface antigen is present on the cells. The cells are brought into good contact with the antibody-coated bottoms of the wells by centrifuging the plates briefly before incubation. Washing is performed by simply immersing the microtiter plates in PBS and flicking off nonadherent cells. The remaining cells are then quantitated using the chromogenic substrate for hexosarninidase. The characteristics of the method are demonstrated in Figs. 6-9. The standard procedure as described in Materials and Methods section was varied. In the experiments in Fig. 6 the concentration and time of coating with the goat anti-mouse I g G antibody was varied. An antibody concentration of 100/~g/ml was chosen as a result. At this concentration, only a small fraction of the protein was lost during each incubation and the solution could therefore be reused. The concentration of monoclonal antibody required for efficiently binding the cells was dependent upon the time allowed for their binding to the anti-mouse Ig.
386
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Fig. 6. Dependency on time of incubation and concentration of goat anti-mouse lg antibody was determined for the binding of Jurcat cells to 3 C l l monoclonal antibodies added at 1/10. Coating with anti-mouse Ig was for 6 h (O), 1½ h (I-n), 20 min (A) or 5 min ( ~ ) . Data represent means of triplicates _+SD. Fig. 7. The indicated concentrations of OKT 9 monoclonal antibodies were allowed to bind to goat anti-mouse Ig coated microtiter plates for 6 h (O), 1½ h (zx), 20 min (1:3) or 5 min (O). The binding of Jurcat cells to the plate was monitored via the hexosaminidase reaction. Means of triplicate samples are given + SD.
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Fig. 8. The effect of adding different numbers of Jurcat cells to microtiter plates coated with goat anti-mouse Ig and the 3Cll monoclonal antibody at 1/10 dilution. The cells were allowed to bind for 4 h ( O , e), 1 h ([3, i ) or 20 min ( ~ , . ) . Before incubation, samples denoted by open symbols had been briefly centrifuged. The amount of chromogenic substrate degraded during 2 h was measured in triplicate. Fig. 9. The stability of monolayer of cells on repeated washing was assessed by allowing Jurcat cells to bind to microtiter wells coated with goat anti-mouse Ig and the 3 C l l hybridoma supernatant at dilutions of 1 / 8 (©), 1/32 (D), 1/128 (~,), 1/512 (zx), or no monoclonal antibody (×). The plates were washed by immersing in a beaker the indicated number of times. Means of triplicates are represented + SD.
387 With a 6 h incubation, concentrations of monoclonal antibody above 0.4 / t g / m l gave plateau levels of cell binding (Fig. 7). Fig. 8 shows the advantage of centrifuging the microtiter plates in shortening the incubation period of cells required. The assay can be performed over a range of cell numbers with good results. One hour of incubation of cells in the cold was found to be satisfactory for ensuring binding to the monoclonal antibodies. The cell monolayer was reasonably stable over a number of washes in wells coated with monoclonal antibody. In contrast, after 3 washes practically no cells remained in wells without antibody (Fig. 9).
Discussion
Enzymes are increasingly being put to use as markers in immune reactions with sensitivities similar to those obtained with radioisotopes. The advantages of employing an ubiquitous lysosomal enzyme, hexosaminidase, in the measurement of cells were first demonstrated by Koponen et al. (1980). In the present study the technique has been characterized and applied to a number of common cellular assays. The method readily lends itself to measurement of cell numbers, provided identical cell types are compared with a standard. With an incubation time of 16 h as few as 800 CTLL-2 cells were detected. The enzyme is quite stable; it resists freezing of cells for a number of days without any effect upon activity. Furthermore, the activity is constant over time even when the incubation time is extended to 16 h. A suitable incubation time may therefore be selected by prolonging incubation in relation to the absorbancy seen in a prematurely developed reaction well. Serum contains high levels of hexosaminidase (Banerjee and Basu, 1975). One microliter of fetal calf serum has the same activity as 3 x 10 4 CTLL-2 cells (data not shown). For this reason it is preferable to wash away remaining serum before the enzyme assay, as was done for the measurement of T C G F or interferon. While the development of immunoassays for T C G F is awaited, it is useful to have a convenient and precise means of quantitating the growth factor. The enzyme assay differs from the thymidine incorporation assay in that it takes into account the cell expansion occurring during the period of the assay, taking account of losses. In contrast, with tritiated thymidine, only DNA synthesis taking place at the time of the pulse is estimated. However, similar sensitivities were obtained by the 2 methods. An approach similar to that described here was recently reported by Nilsson et al. (1983), who estimated cell proliferation in response to T C G F by staining the ceils with Giemsa and then determined the absorbance of the eluted dye. Effector molecules being routinely analyzed include interferons. The virus-protection assay can be made to provide numerical results by assaying for cell survival through measurement of hexosaminidase. It has not, however, proved possible to use measurement of this enzyme to the estimation of survivors after short term cytotoxic assay such as complement mediated |ysis. Cells that have been lysed by the criteria of not excluding trypan blue or retaining 51Cr, still possess the same amount of enzyme activity, even after repeated washings (data not shown).
388
Binding of cells to ligand-coated microtiter wells was used in 2 contexts where the advantages of investigating a large number of samples simultaneously and in small volumes were combined with the possibility of analyzing tests directly in the reaction wells. Firstly, the specific adhesion of hepatocytes to fibronectin was analyzed with findings similar to previously reported results obtained by other methods (Rubin et al., 1979). Secondly, by allowing anti-mouse Ig antibodies to bind to microtiter wells, immunomatrices of the desired specificity could be constructed by adding supernatants containing monoclonal antibodies to the wells. Cells binding to the wells were quantitated through their content of hexosaminidase. This method serves as a sensitive and practical means of analyzing cell populations for the expression of surface antigens as well as for determining the ability of monoclonal antibodies to bind to a given cell type. We have applied it to several cell lines and normal lymphoid cell populations with many different monoclonal antibodies. Currently, it is being used to screen hybridomas producing antibodies with specificity for cell surface antigens. The technique of measuring cells via their content of hexosaminidase as presented in this study is applicable to a sizeable fraction of assays performed in cellular immunology laboratories. The methods are rapid, sensitive and inexpensive. Clearly, many other assays could be adapted to similar measurements.
Acknowledgements I am indepted to Dr. Ulf Heyman for the statistical analysis, to Drs. Gunnar Alm and Kristofer Rubin for help with the interferon and hepatocyte adhesions assays respectively and to Drs. Jan Andersson, Thomas Borg and Hans Wigzell for valuable advice. This study was supported by grants from the Swedish Medical Research Council to Gunnar Aim and Jan Andersson and N I H Grant 5R01-CA26782-04 as well as a grant from the Swedish Cancer Society to Hans Wigzell.
References Applin, J.D. and R.C. Hughes, 1982, Biochim. Biophys. Acta 694, 375. Baker, P.E., S. Gillis and K.A. Smith, 1979, J. Exp. Med. 149, 273. Banerjee, D.K. and D. Basu, 1975, Biochem. J. 145, 113. Gillis, S. and J. Watson, 1980, J. Exp. Med. 152, 1709. Koponen, M., A. Grieder and F. Loor, 1982, Exp. Cell Res. 140, 237. Nilsson, K., W. Scheirer, O.W. Merten, L. Ostberg, E. Liehl, H.W.D. Katinger and K. Mosbach, 1983, Nature (London) 302, 629. Rrnnblom, L. and G.V. Aim, 1982, Eur. J. lmmunol. 12, 437. Rubin, K., S. Johansson, I. Pettersson, C. Ocklind, B. Obrink and M. HrOk, 1979, Biochem. Biophys. Res. Commun. 91, 86. Sutherland, R., D. Delia, C. Schneider, R. Newman, J. Kemshead and M.. Greaves, 1981, Proc. Natl. Aead. Sci. U.S.A. 78, 4515. Wysocki, L.J. and V.L. Sato, 1978, Proc. Natl. Acad. Sci. U.S.A. 75, 2844.
379
Elsevier JIM03011
Measurement of Cell Numbers by Means of the Endogenous Enzyme Hexosaminidase. Applications to Detection of Lymphokines and Cell Surface Antigens Ulf Landegren Department of lmmunology, Biomedical Center, Uppsala University, Box 582, S-751 23 Uppsala, Sweden
(Received 27 June 1983, accepted 1 December 1983)
By using a chromogenic substrate for an ubiquitous lysosomal enzyme, hexosaminidase to estimate cell numbers, a sensitive and simple procedure has been developed in which microtiter reaction wells are directly scanned in a spectrophotometer. This method has been adapted to several cell biological assays. Quantitation of the biological activities of T cell growth factor and interferon can be performed on large numbers of samples. Adhesion of dispersed solid tissue cells to fibronectin coated substrates may be quantitated with little expenditure of reagents. By use of a panning procedure in microtiter plates a sensitive and very simple assay for the binding of monoclonal antibodies to cell surface antigens has been developed. Key words: cell quantitation - growth factor assay - cell surface antigens - cell adhesion - endogenous enzyme assay
Introduction The enzyme N-acetyl-fl-D-hexosaminidase (2-acetamido-2-deoxy-fl-D-glucoside acetamidodeoxuglucohydrolase, EC 3.2.1.30) is a lysosomal enzyme thought to be involved in the d e g r a d a t i o n of glycosylated cellular constituents. It has been d e m o n strated in m a n y tissues a n d in different phyla. A p - n i t r o p h e n o l derivatized substrate can be employed for the spectrophotometric q u a n t i t a t i o n of the activity of this enzyme (Banerjee a n d Basu, 1975). Its widespread occurrence a n d its catalytic activity make this enzyme a n ideal indicator for analysis of cell n u m b e r s . It was first used in this way b y K o p o n e n et al. (1982) to score the sensitivity of a series of t h y m o m a cell lines to the growth i n h i b i t o r y action
Abbreviations." hexosaminidase,N-acetyl-fl-D-hexosaminidase;PBS, phosphate-buffered saline; TCGF, T cell growth factor.
0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.
380 of cyclosporin A. Measurements of this enzyme in cell populations heterogeneous with regard to differentiation stage and lineage need to be analyzed with the caveat that different cells may contribute variably to the overall enzyme reaction. With this limitation in mind, a number of assays, where an effect upon cell numbers is sought, may be accommodated to measurement of this enzyme. I describe here its use for the measurement of cell growth, protection from lysis and binding to substrates via cell surface molecules. Each of these assays is analysed by a number of techniques, commonly involving the use of isotopes. The methods described here allow less manipulation of the samples with good exactitude in determination. In addition, the methods are not dependent upon expensive or unstable reagents.
Materials and Methods
Reagents The substrate for hexosaminidase, p-nitrophenol-N-acetyl-fl-D-glucosaminide was obtained from Sigma (St. Louis, MO). Purified human T cell growth factor (TCGF) was generously supplied by Dr. Richard Robb, Glenolden Laboratories, Glenolden, PA. Human a-interferon was provided by Dr. Gunnar Alm, Departments of Immunology and Medical Virology, BMC, Uppsala. Human fibronectin was a gift by Dr. Kristofer Rubin, Department of Immunology, BMC, Uppsala. F(ab)2 fragments of goat anti-mouse IgG was from Cappel, Cochranville, PA. The OKT 9 monoclonal antibody used had specificity for transferrin receptor (Sutherland et al., 1981) and was obtained from Ortho Pharmaceutical, Raritan, NJ. The 3Cll hybridoma was established in this laboratory by Lena Sporrong. It produces antibodies with affinity for human T cells. Cell culture All cultures were performed in RPMI 1640 with 5% fetal calf serum and supplemented with 2 mM L-glutamine, 50 #M 2-mercaptoethanol, 50 I U / m l penicillin and 50/xg/ml streptomycin. The cell lines investigated were Jurcat, a human T leukemia line (Gillis and Watson, 1980), Raft, a Burkitt lymphoma line, CTLL-2, a T cell growth factor (TCGF) dependent mouse line (Baker et al., 1979) and WISH, a human amniotic cell line. Blood was obtained from volunteers. The cells were separated over Ficoll-Hypaque gradients and the peripheral blood lymphocytes at the interface were employed. Hepatocytes were obtained from rat liver by collagenase perfusion (Rubin et al., 1979). The hexosaminidase reaction The substrate for the enzyme hexosaminidase, p-nitrophenol-N-acetyl-fl-D-glucoseaminide, was dissolved at 7.5 mM in 0.1 M citrate buffer, pH 5. The solution was then mixed with an equal volume of 0.5% Triton X-100 in water, aliquoted and
381
stored at - 1 8 ° C . The substrate solution was added in volumes of 60/~l to cells in flat bottom microtiter wells. The plates were then incubated at 37°C in 100% humidity. After a suitable interval, the color reaction was developed and enzyme activity blocked by addition of 50 mM glycine buffer, p H 10.4, containing 5 mM EDTA, 90/tl per well. Absorbances were measured in a Titertek ® multiskan at 405 nm.
Proliferation assay Cells were grown in flat bottom microtiter plates (Nunc, Denmark). For the thymidine incorporation assay, 2 ~tCi [methyl-3H]thymidine was added per well for the last 2 h of culture. The cells were then harvested on glassfiber paper and processed for scintillation counting. For the enzyme assay, culture was terminated by spinning the plates at 2000 rpm for 5 min. The supernatant was removed by rapidly inverting the plate. Phosphate-buffered saline (PBS) was added and the plates were centrifuged. After flicking the plates again, substrate was added and the enzyme reaction was allowed to proceed for 4 h.
Interferon assay The assay was performed according to R6nnblom and Alm (1982). Briefly, WISH cells were grown in monolayers and exposed to dilutions of interferon. Virus was then added to the wells and culture continued. At the end of incubation, the cells were fixed and stained with crystal violet to determine percentage protection by examination with an inverted microscope. Alternatively, the substrate for hexosaminidase was added and the color reaction measured after 45 min incubation.
Substrate adhesion of hepatocytes Adhesion of hepatocytes was measured by incubating cells for 1 h at 37°C in microtiter wells that had been coated with fibronectin or bovine serum albumin for 1 h at 37°C (Rubin et al., 1979). The wells were washed by immersing the plates in buffer and flicking off unbound cells. After 2 washes, substrate was added for quantitation of cells remaining in the wells.
Detection of antibody binding to cell surface antigens Flat bottom microtiter plates were coated with F(ab)2 fragments of affinity purified goat anti-mouse IgG, 100/~g/ml in 0.05 M Tris buffer, pH 9.5, for 1 h at 37°C. Forty/~l were added per well. The plates were washed 3 times with PBS before the addition 30/~l/well of monoclonal antibodies in medium. Usually a concentration of 1/~g/ml or a 1/10 dilution of hybridoma supernatant was used. After 1½ h at 37°C the plates were washed again. Cells to be tested for adherence to monoclonal antibody coated plates were added in 100/xl, 2 × 105 per well. Hates were centrifuged at 500 rpm for 2 rnin and then incubated for 1½ h at 4°C. At the end of incubation, the plates were washed by immersing in cold PBS and flicking off buffer. Usually no non-specifically adherent cells remained after 3 cycles of washes. This was monitored in an inverted microscope. Quantitation of cells was performed by incubating with substrate and determining absorbancy.
382
Results
The absorbancy of the product of the hexosaminidase reaction is directly proportional to cell numbers and the time allowed for the reaction For analysis of hexosaminidase content, cells were added to microtiter plates in serial dilutions. The plates were centrifuged and the supernatant removed. Substrate was added and the absorbancy of the colored product of the enzyme reaction was determined. In Fig. 1 the 3 lymphocyte lines, CTLL-2, Jurcat and Raji are seen to express similar amounts of enzyme activity. Peripheral blood lymphocytes contain half as much and hepatocytes 5 times as much activity on a per cell basis. CTLL-2 cells were titered in 4 replicate plates and were then exposed to the substrate for variable periods of time. In Fig. 2 the absorbancy is seen to be directly proportional to the number of cells in the well. Furthermore, absorbancy is linearly dependent upon the time allowed for the reaction. When the time for the enzyme reaction was increased 4-fold, one fourth the number of cells are sufficient to give similar absorbancy. One of the plates was stored at - 18°C for 2 days before the 4 h enzyme reaction was carried out. This did not measureably affect enzyme activity. When the results shown in Figs. 1 and 2 are approximated to straight lines, the mean slope is 0.988 + 0.029 SD with a mean correlation coefficient 0.998 + 0.001 SD, indicating direct proportionality between cell numbers and absorbancy. In the calculations, absorbancies between 0.050 and 2.0 have been employed. This allows determination of cell numbers over a 40-fold range for a given time of exposure to the substrate.
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Fig. 1. Hepatocytes (O), CTLL-2 cells (13), Jurcat cells (zx), Raji cells (O), and peripheral blood lymphocytes ( * ) were diluted 2-fold in triplicate in microtiter wells. After removing the supernatant, the substrate for hexosaminidase was added and the plates incubated for 2 h at 37°C. The color reaction was developed and the absorbance determined after blanking the machine on wells that did not contain cells. Points are + SD.
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CELLSI WEll Fig. 2. CTLL-2 cells were diluted 2-fold in quadruplicate in 4 replica microtiter plates. The enzyme was allowed to work on the substrate for 1, 4 or 16 b before the reaction was blocked. A fourth plate was stored at - 1 8 ° C for 2 days before being incubated with substrate for 4 h. The absorbancies of the individual wells are represented by circles. The results have been fitted to straight lines and are shown with confidence intervals ( P < 0.05). Frozen samples are represented by filled circles and are approximated with the dashed line.
Measurement of T cell growth factor activity by quantitation of cells When TCGF is estimated by its ability to induce DNA synthesis in TCGF dependent cells, optimal results are obtained if the thymidine pulse is given 24 h after initiating the culture. At this time any incorporation of tritiated thymidine is 1.5
150
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o 1,0
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2-10 2-9 2-8 2-7 2-6 2-5 2-/, 2-3 2-2 2-1 20 DILUTIONSOFTCSF
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Fig. 3. Four × 104 CTLL-2 cells/ml in 2-fold dilutions of T C G F from 2.5 U / m l were cultured for 24 h before being assayed for incorporation of tritiated thymidine during 2 h (El), or for 48 h before being washed once and then exposed to the substrate for hexosaminidase for 4 h. Absorbancy was determined after blanking the machine on wells that did not receive T C G F (O). The means and SDs of triplicate determinations are shown.
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Fig. 4. WISH cells treated with the indicated concentrations of interferon were challenged with virus at dilutions of 1/400 (t3), 1/1600 (~), 1/6400 (zx) or with no virus (O). Replicate microtiter plates were then (A) assayed for hexosaminidase after 45 min incubation with substrate or (B) fixed and stained and visually scored for protection of individual wells in an inverted microscope. Means of triplicates are given _+SD.
dependent upon T C G F present in the assay while little T C G F will have been consumed. If instead of measuring D N A synthesis, the T C G F dependent survival and expansion of cells is investigated via the hexosaminidase assay, this is optimally done after 48 h of culture. In Fig. 3 results obtained by thymidine incorporation and by the hexosaminidase assay are compared. The sensitivity is similar, but the enzyme assay is more precise and less laborious.
Quantitation of interferon by monitoring hexosaminidase actioity Interferons are estimated by observing their ability to protect virally challenged cells from lysis. This is commonly scored by micro- or macroscopically inspecting the stained cells for protection at the end of the assay. In Fig. 4 results by this method are compared with those by the alternative strategy of quantitating hexosaminidase in cells remaining at the end of culture. Interferon was titered against different doses of virus. While both measurements demonstrate the effect of interferon, the hexosaminidase measurements gives greater variability. In addition a weak cytopathic effect observed at 12.5 I U interferon/ml could not be demonstrated spectrophotometrically. The advantage of the enzyme technique, however, lies in its objectivity and rapidity.
Determination of the ability of hepatocytes to bind to immobilized fibronectin Solid tissue cells carry surface molecules enabling them to adhere to substrates coated with extraceUular matrix macromolecules such as collagen or fibronectin (Applin and Hughes, 1982). The hexosaminidase reaction allows convenient quantitation of adhering cells. In Fig. 5 results are given of an experiment where 2 concentrations of hepatocytes were allowed to settle in wells previously coated with
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Fig. 5. Adhesion of hepatocytes to wells coated with the indicated concentrations of fibronectin (O, e) or bovine serum albumin (O, I). Three× 105 (O, D) or 1 × 105 (O, II) cells were added per well. After the plates had been washed twice, the substrate for hexosaminidase was added for 1½ h. The symbols represent mear.___SD of duplicate determinations.
fibronectin or bovine serum albumin. Concentrations of fibronectin from 10 to 250 /~g/ml permitted stable binding to the microtiter wells. These figures are in accordance with published data (Rubin et al., 1979). Demonstration of cells binding to monoclonal antibodies immobilized in microtiter wells Several techniques are used for analysis of monoclonal antibodies binding to cell surface antigens. A method which combines simplicity and sensitivity with the possibility of avoiding fixing the cells would be valuable for developing hybridomas as well as for investigation of cell surface antigens. For this reason the panning method of Wysocki and Sato (1978) was adapted to microtiter plates. If purified anti-mouse immunoglobulin antibodies are allowed to bind to the wells, cell specific monoclonal antibodies, when bound by the anti-mouse immunoglobulin, will effectively immobilize cells added to the wells, provided the cell surface antigen is present on the cells. The cells are brought into good contact with the antibody-coated bottoms of the wells by centrifuging the plates briefly before incubation. Washing is performed by simply immersing the microtiter plates in PBS and flicking off nonadherent cells. The remaining cells are then quantitated using the chromogenic substrate for hexosarninidase. The characteristics of the method are demonstrated in Figs. 6-9. The standard procedure as described in Materials and Methods section was varied. In the experiments in Fig. 6 the concentration and time of coating with the goat anti-mouse I g G antibody was varied. An antibody concentration of 100/~g/ml was chosen as a result. At this concentration, only a small fraction of the protein was lost during each incubation and the solution could therefore be reused. The concentration of monoclonal antibody required for efficiently binding the cells was dependent upon the time allowed for their binding to the anti-mouse Ig.
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Fig. 6. Dependency on time of incubation and concentration of goat anti-mouse lg antibody was determined for the binding of Jurcat cells to 3 C l l monoclonal antibodies added at 1/10. Coating with anti-mouse Ig was for 6 h (O), 1½ h (I-n), 20 min (A) or 5 min ( ~ ) . Data represent means of triplicates _+SD. Fig. 7. The indicated concentrations of OKT 9 monoclonal antibodies were allowed to bind to goat anti-mouse Ig coated microtiter plates for 6 h (O), 1½ h (zx), 20 min (1:3) or 5 min (O). The binding of Jurcat cells to the plate was monitored via the hexosaminidase reaction. Means of triplicate samples are given + SD.
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Fig. 8. The effect of adding different numbers of Jurcat cells to microtiter plates coated with goat anti-mouse Ig and the 3Cll monoclonal antibody at 1/10 dilution. The cells were allowed to bind for 4 h ( O , e), 1 h ([3, i ) or 20 min ( ~ , . ) . Before incubation, samples denoted by open symbols had been briefly centrifuged. The amount of chromogenic substrate degraded during 2 h was measured in triplicate. Fig. 9. The stability of monolayer of cells on repeated washing was assessed by allowing Jurcat cells to bind to microtiter wells coated with goat anti-mouse Ig and the 3 C l l hybridoma supernatant at dilutions of 1 / 8 (©), 1/32 (D), 1/128 (~,), 1/512 (zx), or no monoclonal antibody (×). The plates were washed by immersing in a beaker the indicated number of times. Means of triplicates are represented + SD.
387 With a 6 h incubation, concentrations of monoclonal antibody above 0.4 / t g / m l gave plateau levels of cell binding (Fig. 7). Fig. 8 shows the advantage of centrifuging the microtiter plates in shortening the incubation period of cells required. The assay can be performed over a range of cell numbers with good results. One hour of incubation of cells in the cold was found to be satisfactory for ensuring binding to the monoclonal antibodies. The cell monolayer was reasonably stable over a number of washes in wells coated with monoclonal antibody. In contrast, after 3 washes practically no cells remained in wells without antibody (Fig. 9).
Discussion
Enzymes are increasingly being put to use as markers in immune reactions with sensitivities similar to those obtained with radioisotopes. The advantages of employing an ubiquitous lysosomal enzyme, hexosaminidase, in the measurement of cells were first demonstrated by Koponen et al. (1980). In the present study the technique has been characterized and applied to a number of common cellular assays. The method readily lends itself to measurement of cell numbers, provided identical cell types are compared with a standard. With an incubation time of 16 h as few as 800 CTLL-2 cells were detected. The enzyme is quite stable; it resists freezing of cells for a number of days without any effect upon activity. Furthermore, the activity is constant over time even when the incubation time is extended to 16 h. A suitable incubation time may therefore be selected by prolonging incubation in relation to the absorbancy seen in a prematurely developed reaction well. Serum contains high levels of hexosaminidase (Banerjee and Basu, 1975). One microliter of fetal calf serum has the same activity as 3 x 10 4 CTLL-2 cells (data not shown). For this reason it is preferable to wash away remaining serum before the enzyme assay, as was done for the measurement of T C G F or interferon. While the development of immunoassays for T C G F is awaited, it is useful to have a convenient and precise means of quantitating the growth factor. The enzyme assay differs from the thymidine incorporation assay in that it takes into account the cell expansion occurring during the period of the assay, taking account of losses. In contrast, with tritiated thymidine, only DNA synthesis taking place at the time of the pulse is estimated. However, similar sensitivities were obtained by the 2 methods. An approach similar to that described here was recently reported by Nilsson et al. (1983), who estimated cell proliferation in response to T C G F by staining the ceils with Giemsa and then determined the absorbance of the eluted dye. Effector molecules being routinely analyzed include interferons. The virus-protection assay can be made to provide numerical results by assaying for cell survival through measurement of hexosaminidase. It has not, however, proved possible to use measurement of this enzyme to the estimation of survivors after short term cytotoxic assay such as complement mediated |ysis. Cells that have been lysed by the criteria of not excluding trypan blue or retaining 51Cr, still possess the same amount of enzyme activity, even after repeated washings (data not shown).
388
Binding of cells to ligand-coated microtiter wells was used in 2 contexts where the advantages of investigating a large number of samples simultaneously and in small volumes were combined with the possibility of analyzing tests directly in the reaction wells. Firstly, the specific adhesion of hepatocytes to fibronectin was analyzed with findings similar to previously reported results obtained by other methods (Rubin et al., 1979). Secondly, by allowing anti-mouse Ig antibodies to bind to microtiter wells, immunomatrices of the desired specificity could be constructed by adding supernatants containing monoclonal antibodies to the wells. Cells binding to the wells were quantitated through their content of hexosaminidase. This method serves as a sensitive and practical means of analyzing cell populations for the expression of surface antigens as well as for determining the ability of monoclonal antibodies to bind to a given cell type. We have applied it to several cell lines and normal lymphoid cell populations with many different monoclonal antibodies. Currently, it is being used to screen hybridomas producing antibodies with specificity for cell surface antigens. The technique of measuring cells via their content of hexosaminidase as presented in this study is applicable to a sizeable fraction of assays performed in cellular immunology laboratories. The methods are rapid, sensitive and inexpensive. Clearly, many other assays could be adapted to similar measurements.
Acknowledgements I am indepted to Dr. Ulf Heyman for the statistical analysis, to Drs. Gunnar Alm and Kristofer Rubin for help with the interferon and hepatocyte adhesions assays respectively and to Drs. Jan Andersson, Thomas Borg and Hans Wigzell for valuable advice. This study was supported by grants from the Swedish Medical Research Council to Gunnar Aim and Jan Andersson and N I H Grant 5R01-CA26782-04 as well as a grant from the Swedish Cancer Society to Hans Wigzell.
References Applin, J.D. and R.C. Hughes, 1982, Biochim. Biophys. Acta 694, 375. Baker, P.E., S. Gillis and K.A. Smith, 1979, J. Exp. Med. 149, 273. Banerjee, D.K. and D. Basu, 1975, Biochem. J. 145, 113. Gillis, S. and J. Watson, 1980, J. Exp. Med. 152, 1709. Koponen, M., A. Grieder and F. Loor, 1982, Exp. Cell Res. 140, 237. Nilsson, K., W. Scheirer, O.W. Merten, L. Ostberg, E. Liehl, H.W.D. Katinger and K. Mosbach, 1983, Nature (London) 302, 629. Rrnnblom, L. and G.V. Aim, 1982, Eur. J. lmmunol. 12, 437. Rubin, K., S. Johansson, I. Pettersson, C. Ocklind, B. Obrink and M. HrOk, 1979, Biochem. Biophys. Res. Commun. 91, 86. Sutherland, R., D. Delia, C. Schneider, R. Newman, J. Kemshead and M.. Greaves, 1981, Proc. Natl. Aead. Sci. U.S.A. 78, 4515. Wysocki, L.J. and V.L. Sato, 1978, Proc. Natl. Acad. Sci. U.S.A. 75, 2844.