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Concanavalin a binding material derived from a tissue culture medium
© 1972 by Academic Press, Inc.
J. ULTRASTRUCTUR~RESEARCI~40, 145-151 (1972)
145
Concanavalin A Binding Material Derived from a Tissue Culture Medium CHARLES ROWLATT1 and REN~ WICKER
Unitks de Microscopic Eleetronique et de Virologie, Institut de Recherches Seientifiques sur le Cancer, 94-Villejuif, France Received January 21, 1972 Material to which concanavalin A bound was observed on the surfaces of bottles that had been treated with a complete tissue culture medium. In one series of experiments, the bound concanavalin A was demonstrated by the adherence of suspended hamster cells. In the second series, the presence of the concanavalin A binding material on the surface of the culture bottles was demonstrated by ultrastructural cytochemistry. It was present after treatment with the culture medium, whether or not hamster cells had been grown. Apparently striking differences in the agglutinability of suspensions of transformed and nontransformed cell lines using plant agglutinins (6, 11) has led to much work with these materials. However, reports are now appearing which show that there is considerable variation between cell types (7, 13) and that the phenomena may change with time in culture (8). We have found another problem in the interpretation of experiments in which agglutinins are applied to cells in tissue culture, in controls in two separate series of experiments. Material to which concanavalin A binds is also present on culture surfaces and appears to be derived from the tissue culture medium itself. EXPERIMENTAL CONDITIONS AND RESULTS In the first series, the attachment of different cells to the wall of Falcon flasks was studied with and without pretreatment with concanavalin A. We used nonmalignant cells [embryo hamster secondary cultures (EH)] and three hamster tumor cell lines [T PY XV (Py) (5); C12 TSV5 (SV40) (•7); T A d l 2 XIV (Adl2) (5)] which were grown for 48 hours in Eagle's M E M medium with 10 % calf serum and 10 % tryptose phosphate (Difco). Cells were separated with E D T A [0.02 % disodium ethylene diaminotetraacetate in Dulbecco's phosphate-buffered saline with calcium and mag1 Permanent address: Department of Cellular Pathology, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, England. 10-- 721833 J . Ultrastructure Research
146
ROWLATT AND WICKER TABLE I
RESULTS OF 8 EXPERIMENTS COMPARING THE ADHERENCE TO THE SURFACE OF FALCON FLASKS OF VARIOUS CELL TYPES AFTER DIFFERENT TREATMENTSa Contact with medium None None None None None 3 Hours 24 Hours 24 Hours 3½ Hours 3½ Hours 48 Hours 48 Hours 1¼ Hours 11 Hours 7 Days 7 Days
Cell type EH Py EH Py Et-I EH EH Py EH SV40 EH SV40 EH Adl2 EH Adl2
Pretreatment with ~-" -~" PBS ConA + ++/+++ + + + + + + O +/++ o/+ o/+ + + O/+ + + + O/+ +
++/+++ ++/+++ + + + + + + ~ + J ++++ + + + + + + + + + + + + + + + + + + + + + + + + + + +
Notesb ~ J
1,2
3 ~ J
4
~ J
4, 5
Although cells may adhere to the surface of flasks which have not been in contact with complete medium, this adherence is normally unaffected by immediate pretreatment with concanavalin A (conA). ConA causes a marked increase in the adherence of cells of all types to flasks which have contained medium. Adherence was assessed on a scale from © to + + + + by each author independently. Key to notes: (1) Suspended cells marked with 0.01% neutral red in medium 1 hour before separation. (2) Cells used at 72 hours. (3) Double rinse before final fixation. (4) Cells marked with India ink after method of Stoker (15)--carbon added to medium for first 24 hours of growth, but without extra cleaning with trypsin. (5) Illustrated in Fig. 1.
n e s i u m o m i t t e d (PBS)], w a s h e d 3 t i m e s w i t h P B S at 4°C, a n d r e s u s p e n d e d at t h e w o r k i n g c o n c e n t r a t i o n . I n the c o n t r o l w h i c h c o n c e r n s us here, p o l y s t y r e n e flasks ( F a l c o n ) w e r e i n c u b a t e d f o r v a r i o u s times, w i t h a n d w i t h o u t the c o m p l e t e tissue cult u r e m e d i u m (as a b o v e ) , c o o l e d to r o o m t e m p e r a t u r e (25-27°C), r i n s e d t w i c e w i t h PBS, t r e a t e d w i t h c o n c a n a v a l i n A [purified (1) f r o m J a c k b e a n m e a l , Sigma, by S. A v r a m e a s ] 50 # g / m l in PBS, o r PBS, f o r 3 m i n u t e s at r o o m t e m p e r a t u r e , r i n s e d o n c e w i t h P B S b e f o r e t h e a d d i t i o n of 3 to 5 x 106 cells in 5 m l of P B S at r o o m t e m p e r a ture. A f t e r 5 m i n u t e s , the cell s u s p e n s i o n was t i p p e d off in a s t a n d a r d m a n n e r a n d t h e flasks w e r e r i n s e d o n c e in P B S b e f o r e f i x a t i o n of a n y a d h e r e n t cells in 10 % f o r m a l i n f o r 10 m i n u t e s . T h e flasks w e r e d r a i n e d a n d the n u m b e r of a d h e r e n t cells were e v a l u a t e d ( T a b l e I) by p h a s e c o n t r a s t m i c r o s c o p y (Leitz). I n three r u n s t h e s u s p e n d e d cells h a d b e e n vitally stained f o r o t h e r p u r p o s e s . m o u n t e d in E l v a n o l or glycerol.
Some specimens were
CONCANAVALIN A BINDING MATERIAL IN CULTURE MEDIUM
147
FIG. 1. Phase contrast photomicrographs of the floor of Falcon tissue culture flasks, which had been incubated with complete tissue culture medium for 1¼ hours, to show the attachment of vitally stained suspended cells which had been in contact with the surface for 5 minutes. Scale line = 50/~. (a) EH cells: pretreatment of flask with PBS. (b) EH cells: pretreatment with concanavalin A. (c) ADI2 cells: pretreatment with PBS. (d) AD12 cells: pretreatment with concanavalin A.
Table I shows that, when flasks w i t h o u t m e d i u m were used, cells a d h e r e d to the surface, b u t i m m e d i a t e p r e t r e a t m e n t with c o n c a n a v a l i n A did n o t increase this in m o s t cases. I n contrast, when the flasks h a d been i n c u b a t e d first with m e d i u m , a m a r k e d increase in cellular adherence was seen after c o n c a n a v a l i n t r e a t m e n t (illustrated in Fig. 1). Vital staining of the cells a p p e a r e d to increase their a t t a c h m e n t to the plastic. F r o m this e x p e r i m e n t we conclude t h a t p r e t r e a t m e n t of the flasks with m e d i u m c o a t e d the culture surface with m a t e r i a l to which c o n c a n a v a l i n A b o u n d ,
148
ROWLATT AND WICKER
and that this concanavalin A, which was not washed off, caused the untreated cells to adhere more firmly. In the second series of experiments, the distribution of concanavalin A binding material in the cell-free areas of 60 ml glass medicine bottles containing 48-hour cultures of EH and Adl2, and in the same bottles treated with culture medium without cells, was examined in the electron microscope. The localization was by Avrameas' method (3) developed for ultrastructural cytochemistry by Bernhard and Avrameas (4): horseradish peroxidase is used to mark the concanavalin attached to specific sugars or glycoproteins, and the enzyme was localized, after fixation, by the method of Graham and Karnovsky (10). The cultures were allowed to cool and were rinsed 3 times with PBS before treatment with concanavalin A (solution as above) with or without 0.1 M glucose (as a specific blocking agent) for 15 minutes at room temperature. After three rinses with PBS, the cultures were treated with horseradish peroxidase (Sigma type VI, 50 #g/ml in PBS) or PBS for 15 minutes. After three further PBS rinses, the cultures were fixed in 1.6 % glutaraldehyde in PBS for 15 minutes. The peroxidase was revealed by diaminobenzidine and postosmication (4, 10) before ethanol dehydration, propylene oxide, and Epon embedding, all performed on the cells in situ. EH and Adl2 cultures were also fixed with glutaraldehyde (2.5 % in cacodylate buffer) with and without 0.05 % ruthenium red (Fluka), for 1 hour at 4°C in the dark, followed, after washing with buffer, by OsO~ fixation (2 % in cacodylate buffer) for 3 hours in the dark with and without ruthenium red. All cultures were sectioned in a vertical plane after the Epon had been separated from the glass. It is found that, when the surface formerly attached to the glass is supported with a secondary layer of Epon, surface material from the culture surface is preserved and can be seen in the electron microscope by conventional methods (Fig. 2a). In view of the findings in the first series of experiments, this layer was studied more closely. Concanavalin A attaches to this culture surface material, and a positive reaction (Fig. 2b)was found in all 10 cultures (5 x EH, 5 x Adl2) examined. This reaction was blocked by glucose added to the concanavalin solution (Fig. 2c), although in some areas staining attributable to nonspecific peroxidase activity from broken down cells could be found (Fig. 2 d). The concanavalin-positive material normally extended over the whole culture surface, but in some cultures it was only found in patches. Material which was concanavalin positive was also found on the surface of a control bottle which had been incubated with complete medium but without cells (Fig. 2e). Glucose blocked this reaction, and when the blocked material was poststained (Fig. 2f) a surface coat very similar to that in the conventional preparations (Fig. 2a) was found. No concanavalin-positive material was found on the surface of the bottle incubated without medium (Fig. 2g).
CONCANAVALIN A BINDING MATERIAL IN CULTURE MEDIUM
149
FIG. 2. Electron micrographs to show the staining reactions of the surface layer on tissue culture bottles (glass). See text for description. Scale line = 1 000 A. x 60 000. (a-d) Cell-free areas in bottles with E H cultures: (a) Glutaraldehyde, osmium, post stain uranium, lead. (b) Concanavalin reaction, no post stain. (c) Concanavalin reaction blocked with 0.1 M glucose. (d) Concanavalin reaction but with peroxidase omitted. (e-f) Bottles with medium but no cells: (e) Concanavalin reaction (no post stain). (f) Concanavalin reaction blocked by glucose, post stain uranium, lead. (g) Bottle alone: Concanavalin reaction, post stain uranium, lead. (h) Cell-free area in bottle with E H culture: ruthenium red with glutaraldehyde and with osmium.
150
ROWLATT AND WICKER
The surface coating on the glass of culture bottles is also positive to ruthenium red (Fig. 2 h), another cell surface mucopolysaccharide stain (12). A layer of material which gives a positive concanavalin A reaction has also been present on the cell-free areas of Falcon flasks, when these have been used for similar cultures (J. M a t o ; k a , personal communication).
DISCUSSION We have presented evidence from two different types of experiment that material to which concanavalin A can bind coats the interior of culture bottles which have been in contact with a complete tissue culture medium. The findings emphasize difficulties in interpreting cell surface studies of cells raised or examined in vitro. Although it has not been demonstrated specifically in this study, it is probable that the surface material is derived from the serum (16). The coating of both normal (16) and tumor (2) cells with serum components has been postulated, and it seems probable that some of the concanavalin-positive material on the surface of cultured cells may be derived from the medium. Another difficulty is in the interpretation of in vitro studies of cell mobility (e.g., 9) or growth (e.g., 14) when concanavalin is added to the medium. If concanavalin binds to the surface of the culture vessels as well as to the cells, it seems extremely likely that this will cause the attachment of the cells to the substrate. This alone could materially alter cell mobility and growth, without necessarily affecting intracellular processes directly. The authors are extremely grateful to M. W. Bernhard for electron microscopy facilities and advice. The observations were made as a consequence of other studies started with Drs A. Martinez-Palomo and R. Bretton.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
AGRAWAL,B. B. L. and GOLDSTEIN,I. J., Biochim. Biophys. Acta 133, 376 (1967). AVFFEL,C. A. and PETERS,J. H., Progr. Exp. Tumor Res. 12, 1 (1969). AVRAMEAS,S., C. R. Acad. Sci. Ser. D 270, 2205 (1970). BERNHARD,W. and AVRAMEAS,S., Exp. Cell Res. 64, 232 (1971). BERNHARD,W. and TOt~RNIER,P., Int. Y. Cancer 1, 61 (1966). BtJR~ER, M. M. and GOLDBERC,A. R., Proc. Nat. Aead. Sci. USA 57, 359 (1967). CLINE, M. J. and LIVINGSTON,D. C., Nature (London) 232, 155 (1971). CRONIN,A. P., BmDLE, F. and SANDERS,F. K., Cytobios 2 (7-8), 225 (1970). FRIBERG,S., COCHRAY, A. J. and GOLUB, S. H., Nature (London) 232, 121 (1971). GRAHAM,R. C. and KARNOVSKY,M. J., J. Histoehem. Cytochem. 14, 291 (1966). INBAR, M. and SACHS, L., Proe. Nat. Acad. Sci. USA 63, 1418 (1969). LuET, J. H., Fed. Proe. Fed. Amer. Soe. Exp. Biol. 25, 1773 (1966).
CONCANAVALINA BINDING MATERIALIN CULTURE MEDIUM 13. 14. 15. 16. 17.
151
MOORE, E. G. and TEMIN, H. M., Nature (London) 231, 117 (1971). POWELL,A. E. and LEON, M. A., Exp. Cell Res. 62, 315 (1970). SXOKER,M., Exp. Cell Res. 35, 429 (1964). TAYLOR,A. C., Exp. Cell Res., Suppl 8, 154 (1961). TOURNIER,P., CASSINGENA,R., WICKER, R., COPPEY, J. and SUAREZ,H. G., Int. J. Cancer 2, 117 (1967).
J. ULTRASTRUCTUR~RESEARCI~40, 145-151 (1972)
145
Concanavalin A Binding Material Derived from a Tissue Culture Medium CHARLES ROWLATT1 and REN~ WICKER
Unitks de Microscopic Eleetronique et de Virologie, Institut de Recherches Seientifiques sur le Cancer, 94-Villejuif, France Received January 21, 1972 Material to which concanavalin A bound was observed on the surfaces of bottles that had been treated with a complete tissue culture medium. In one series of experiments, the bound concanavalin A was demonstrated by the adherence of suspended hamster cells. In the second series, the presence of the concanavalin A binding material on the surface of the culture bottles was demonstrated by ultrastructural cytochemistry. It was present after treatment with the culture medium, whether or not hamster cells had been grown. Apparently striking differences in the agglutinability of suspensions of transformed and nontransformed cell lines using plant agglutinins (6, 11) has led to much work with these materials. However, reports are now appearing which show that there is considerable variation between cell types (7, 13) and that the phenomena may change with time in culture (8). We have found another problem in the interpretation of experiments in which agglutinins are applied to cells in tissue culture, in controls in two separate series of experiments. Material to which concanavalin A binds is also present on culture surfaces and appears to be derived from the tissue culture medium itself. EXPERIMENTAL CONDITIONS AND RESULTS In the first series, the attachment of different cells to the wall of Falcon flasks was studied with and without pretreatment with concanavalin A. We used nonmalignant cells [embryo hamster secondary cultures (EH)] and three hamster tumor cell lines [T PY XV (Py) (5); C12 TSV5 (SV40) (•7); T A d l 2 XIV (Adl2) (5)] which were grown for 48 hours in Eagle's M E M medium with 10 % calf serum and 10 % tryptose phosphate (Difco). Cells were separated with E D T A [0.02 % disodium ethylene diaminotetraacetate in Dulbecco's phosphate-buffered saline with calcium and mag1 Permanent address: Department of Cellular Pathology, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, England. 10-- 721833 J . Ultrastructure Research
146
ROWLATT AND WICKER TABLE I
RESULTS OF 8 EXPERIMENTS COMPARING THE ADHERENCE TO THE SURFACE OF FALCON FLASKS OF VARIOUS CELL TYPES AFTER DIFFERENT TREATMENTSa Contact with medium None None None None None 3 Hours 24 Hours 24 Hours 3½ Hours 3½ Hours 48 Hours 48 Hours 1¼ Hours 11 Hours 7 Days 7 Days
Cell type EH Py EH Py Et-I EH EH Py EH SV40 EH SV40 EH Adl2 EH Adl2
Pretreatment with ~-" -~" PBS ConA + ++/+++ + + + + + + O +/++ o/+ o/+ + + O/+ + + + O/+ +
++/+++ ++/+++ + + + + + + ~ + J ++++ + + + + + + + + + + + + + + + + + + + + + + + + + + +
Notesb ~ J
1,2
3 ~ J
4
~ J
4, 5
Although cells may adhere to the surface of flasks which have not been in contact with complete medium, this adherence is normally unaffected by immediate pretreatment with concanavalin A (conA). ConA causes a marked increase in the adherence of cells of all types to flasks which have contained medium. Adherence was assessed on a scale from © to + + + + by each author independently. Key to notes: (1) Suspended cells marked with 0.01% neutral red in medium 1 hour before separation. (2) Cells used at 72 hours. (3) Double rinse before final fixation. (4) Cells marked with India ink after method of Stoker (15)--carbon added to medium for first 24 hours of growth, but without extra cleaning with trypsin. (5) Illustrated in Fig. 1.
n e s i u m o m i t t e d (PBS)], w a s h e d 3 t i m e s w i t h P B S at 4°C, a n d r e s u s p e n d e d at t h e w o r k i n g c o n c e n t r a t i o n . I n the c o n t r o l w h i c h c o n c e r n s us here, p o l y s t y r e n e flasks ( F a l c o n ) w e r e i n c u b a t e d f o r v a r i o u s times, w i t h a n d w i t h o u t the c o m p l e t e tissue cult u r e m e d i u m (as a b o v e ) , c o o l e d to r o o m t e m p e r a t u r e (25-27°C), r i n s e d t w i c e w i t h PBS, t r e a t e d w i t h c o n c a n a v a l i n A [purified (1) f r o m J a c k b e a n m e a l , Sigma, by S. A v r a m e a s ] 50 # g / m l in PBS, o r PBS, f o r 3 m i n u t e s at r o o m t e m p e r a t u r e , r i n s e d o n c e w i t h P B S b e f o r e t h e a d d i t i o n of 3 to 5 x 106 cells in 5 m l of P B S at r o o m t e m p e r a ture. A f t e r 5 m i n u t e s , the cell s u s p e n s i o n was t i p p e d off in a s t a n d a r d m a n n e r a n d t h e flasks w e r e r i n s e d o n c e in P B S b e f o r e f i x a t i o n of a n y a d h e r e n t cells in 10 % f o r m a l i n f o r 10 m i n u t e s . T h e flasks w e r e d r a i n e d a n d the n u m b e r of a d h e r e n t cells were e v a l u a t e d ( T a b l e I) by p h a s e c o n t r a s t m i c r o s c o p y (Leitz). I n three r u n s t h e s u s p e n d e d cells h a d b e e n vitally stained f o r o t h e r p u r p o s e s . m o u n t e d in E l v a n o l or glycerol.
Some specimens were
CONCANAVALIN A BINDING MATERIAL IN CULTURE MEDIUM
147
FIG. 1. Phase contrast photomicrographs of the floor of Falcon tissue culture flasks, which had been incubated with complete tissue culture medium for 1¼ hours, to show the attachment of vitally stained suspended cells which had been in contact with the surface for 5 minutes. Scale line = 50/~. (a) EH cells: pretreatment of flask with PBS. (b) EH cells: pretreatment with concanavalin A. (c) ADI2 cells: pretreatment with PBS. (d) AD12 cells: pretreatment with concanavalin A.
Table I shows that, when flasks w i t h o u t m e d i u m were used, cells a d h e r e d to the surface, b u t i m m e d i a t e p r e t r e a t m e n t with c o n c a n a v a l i n A did n o t increase this in m o s t cases. I n contrast, when the flasks h a d been i n c u b a t e d first with m e d i u m , a m a r k e d increase in cellular adherence was seen after c o n c a n a v a l i n t r e a t m e n t (illustrated in Fig. 1). Vital staining of the cells a p p e a r e d to increase their a t t a c h m e n t to the plastic. F r o m this e x p e r i m e n t we conclude t h a t p r e t r e a t m e n t of the flasks with m e d i u m c o a t e d the culture surface with m a t e r i a l to which c o n c a n a v a l i n A b o u n d ,
148
ROWLATT AND WICKER
and that this concanavalin A, which was not washed off, caused the untreated cells to adhere more firmly. In the second series of experiments, the distribution of concanavalin A binding material in the cell-free areas of 60 ml glass medicine bottles containing 48-hour cultures of EH and Adl2, and in the same bottles treated with culture medium without cells, was examined in the electron microscope. The localization was by Avrameas' method (3) developed for ultrastructural cytochemistry by Bernhard and Avrameas (4): horseradish peroxidase is used to mark the concanavalin attached to specific sugars or glycoproteins, and the enzyme was localized, after fixation, by the method of Graham and Karnovsky (10). The cultures were allowed to cool and were rinsed 3 times with PBS before treatment with concanavalin A (solution as above) with or without 0.1 M glucose (as a specific blocking agent) for 15 minutes at room temperature. After three rinses with PBS, the cultures were treated with horseradish peroxidase (Sigma type VI, 50 #g/ml in PBS) or PBS for 15 minutes. After three further PBS rinses, the cultures were fixed in 1.6 % glutaraldehyde in PBS for 15 minutes. The peroxidase was revealed by diaminobenzidine and postosmication (4, 10) before ethanol dehydration, propylene oxide, and Epon embedding, all performed on the cells in situ. EH and Adl2 cultures were also fixed with glutaraldehyde (2.5 % in cacodylate buffer) with and without 0.05 % ruthenium red (Fluka), for 1 hour at 4°C in the dark, followed, after washing with buffer, by OsO~ fixation (2 % in cacodylate buffer) for 3 hours in the dark with and without ruthenium red. All cultures were sectioned in a vertical plane after the Epon had been separated from the glass. It is found that, when the surface formerly attached to the glass is supported with a secondary layer of Epon, surface material from the culture surface is preserved and can be seen in the electron microscope by conventional methods (Fig. 2a). In view of the findings in the first series of experiments, this layer was studied more closely. Concanavalin A attaches to this culture surface material, and a positive reaction (Fig. 2b)was found in all 10 cultures (5 x EH, 5 x Adl2) examined. This reaction was blocked by glucose added to the concanavalin solution (Fig. 2c), although in some areas staining attributable to nonspecific peroxidase activity from broken down cells could be found (Fig. 2 d). The concanavalin-positive material normally extended over the whole culture surface, but in some cultures it was only found in patches. Material which was concanavalin positive was also found on the surface of a control bottle which had been incubated with complete medium but without cells (Fig. 2e). Glucose blocked this reaction, and when the blocked material was poststained (Fig. 2f) a surface coat very similar to that in the conventional preparations (Fig. 2a) was found. No concanavalin-positive material was found on the surface of the bottle incubated without medium (Fig. 2g).
CONCANAVALIN A BINDING MATERIAL IN CULTURE MEDIUM
149
FIG. 2. Electron micrographs to show the staining reactions of the surface layer on tissue culture bottles (glass). See text for description. Scale line = 1 000 A. x 60 000. (a-d) Cell-free areas in bottles with E H cultures: (a) Glutaraldehyde, osmium, post stain uranium, lead. (b) Concanavalin reaction, no post stain. (c) Concanavalin reaction blocked with 0.1 M glucose. (d) Concanavalin reaction but with peroxidase omitted. (e-f) Bottles with medium but no cells: (e) Concanavalin reaction (no post stain). (f) Concanavalin reaction blocked by glucose, post stain uranium, lead. (g) Bottle alone: Concanavalin reaction, post stain uranium, lead. (h) Cell-free area in bottle with E H culture: ruthenium red with glutaraldehyde and with osmium.
150
ROWLATT AND WICKER
The surface coating on the glass of culture bottles is also positive to ruthenium red (Fig. 2 h), another cell surface mucopolysaccharide stain (12). A layer of material which gives a positive concanavalin A reaction has also been present on the cell-free areas of Falcon flasks, when these have been used for similar cultures (J. M a t o ; k a , personal communication).
DISCUSSION We have presented evidence from two different types of experiment that material to which concanavalin A can bind coats the interior of culture bottles which have been in contact with a complete tissue culture medium. The findings emphasize difficulties in interpreting cell surface studies of cells raised or examined in vitro. Although it has not been demonstrated specifically in this study, it is probable that the surface material is derived from the serum (16). The coating of both normal (16) and tumor (2) cells with serum components has been postulated, and it seems probable that some of the concanavalin-positive material on the surface of cultured cells may be derived from the medium. Another difficulty is in the interpretation of in vitro studies of cell mobility (e.g., 9) or growth (e.g., 14) when concanavalin is added to the medium. If concanavalin binds to the surface of the culture vessels as well as to the cells, it seems extremely likely that this will cause the attachment of the cells to the substrate. This alone could materially alter cell mobility and growth, without necessarily affecting intracellular processes directly. The authors are extremely grateful to M. W. Bernhard for electron microscopy facilities and advice. The observations were made as a consequence of other studies started with Drs A. Martinez-Palomo and R. Bretton.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
AGRAWAL,B. B. L. and GOLDSTEIN,I. J., Biochim. Biophys. Acta 133, 376 (1967). AVFFEL,C. A. and PETERS,J. H., Progr. Exp. Tumor Res. 12, 1 (1969). AVRAMEAS,S., C. R. Acad. Sci. Ser. D 270, 2205 (1970). BERNHARD,W. and AVRAMEAS,S., Exp. Cell Res. 64, 232 (1971). BERNHARD,W. and TOt~RNIER,P., Int. Y. Cancer 1, 61 (1966). BtJR~ER, M. M. and GOLDBERC,A. R., Proc. Nat. Aead. Sci. USA 57, 359 (1967). CLINE, M. J. and LIVINGSTON,D. C., Nature (London) 232, 155 (1971). CRONIN,A. P., BmDLE, F. and SANDERS,F. K., Cytobios 2 (7-8), 225 (1970). FRIBERG,S., COCHRAY, A. J. and GOLUB, S. H., Nature (London) 232, 121 (1971). GRAHAM,R. C. and KARNOVSKY,M. J., J. Histoehem. Cytochem. 14, 291 (1966). INBAR, M. and SACHS, L., Proe. Nat. Acad. Sci. USA 63, 1418 (1969). LuET, J. H., Fed. Proe. Fed. Amer. Soe. Exp. Biol. 25, 1773 (1966).
CONCANAVALINA BINDING MATERIALIN CULTURE MEDIUM 13. 14. 15. 16. 17.
151
MOORE, E. G. and TEMIN, H. M., Nature (London) 231, 117 (1971). POWELL,A. E. and LEON, M. A., Exp. Cell Res. 62, 315 (1970). SXOKER,M., Exp. Cell Res. 35, 429 (1964). TAYLOR,A. C., Exp. Cell Res., Suppl 8, 154 (1961). TOURNIER,P., CASSINGENA,R., WICKER, R., COPPEY, J. and SUAREZ,H. G., Int. J. Cancer 2, 117 (1967).