1, Chick embryo fibroblasts, that cease to grow after a finite number of passages in culture, have been cultured in the presence of radioactive D-g~ucosaminc. Incorporation of radioactivity into cellular macromolecules is very similar for cells regardless of their position in the finite life span.2. The labelled substances removed by trypsin from the surface of non-confluent cells growing in different parts of the life span were analysed. Smooth membrane fractions isolated from trypsinised cells were also compared.3. The labelled substances were very similar, in particular the extent of sialylation of the total glycoprotein fraction of early phase or late phase cells. Minor differences were found, however.4. It was found that mucopolysaccharides constitute a larger proportion of the labelled material from the surface of late phase cells than from the surface of early phase cells. Conversely early phasc cells were relatively rich in several glycopeptides compared t o latc phase cells. We have begun a study of the membranes of chick embryo fibroblasts undergoing senescence in vitro in an attempt to detect age-related membrane phenomena. I n this paper we report on certain aspects of the glycoproteins and mucopolysaccharides of cells grown to different parts of the life span in culture.
EXPERIMENTAL PROCEDURE
Cell CulturesChick fibroblasts were obtained from leg muscle of 8-to 10-day-old chick embryos. The tissue was minced and disaggregated at 37 "C for 30 min with O.O5O/, trypsin in Ca2+-and Mgz+-free Hank's balanced salts solution.The single cells were recovered by centrifugation a t 800 x g for 4 min after removal of gross tissue fragments by gravity sedimentation, washed with complete medium and counted. Complete medium was Eagles minimal essential medium with loo/, fetal calf serum, penicillin (100 U/ml) and streptomycin (I00 pg/ml). Dispersed cells ( los)were seeded into Falcon roller bottles (growth area of 690 cmz) containing 150 ml medium which were rotated at 0.7 rev./min and kept st 37 "C in a 501, COZ/95O/, air atmosphere. At confluency the cells were removed by treatment at 37 "C for 10 min with O.2ij0/, trypsin in isotonic saline, washed and seeded into two bottles. This operation is referred to as one passage and is repeated until the cells no longer Em. J. Biochem. 44 (1974)