Abstract
Most investigators are cognizant of the problems inherent in counting cells embedded in a complex and abundant extracellular matrix. To overcome these obstacles, we developed a new method of isolating nucleic acids from chondrocytes which facilitates measurement of cell number by DNA analysis. Chondrocytes were isolated from chick embryo sterna and grown continuously without subculturing for 2–3 weeks in monolayer. The cells were treated with triton X-100 and the nucleic acid content of the extract was determined by measuring DNA fluorescence in the presence of Hoechst dye 33258. To minimize background fluorescence due to the triton, we precipitated the DNA with alcohol and then solubilized the nucleic acids in EDTA. This simple procedure removed the detergent and substantially increased the sensitivity of the method. Thus, we could measure with high precision and high recovery, the DNA content of cultures of 10,000–50,000 cells. In a single well containing 0.5–1.0 million cells, sufficient material remained for subsequent measurements of alkaline phosphatase activity and protein and calcium content. As the mineral present in the triton-treated samples was soluble in EDTA, we experienced no problems in measuring the calcium content of the culture. In addition, as triton X-100 is a nonionic detergent, we were able to measure cell and matrix proteins; moreover, the presence of the triton maintained the catalytic state of alkaline phosphatase. We conclude that this procedure provides a simple and rapid approach to measuring major indicators of chondrocyte maturation and function.
References
Stocum DL, Davis RM, Leger M, Conrad HE (1979) Development of the tibiotarsus in the chick embryo: biosynthetic activities of histologically distinct regions. J Embryol Exp Morphol 54:155–170
Hunzinger EB, Schenk RK, Cruz-Orive L (1987) Quantitation of chondrocyte performance in growth plate cartilage during longitudinal bone growth. J Bone Jt Surg 69A:162–173
Schmid TM, Linsenmayer TF (1985) Immunohistochemical localization of short chain cartilage collagen (type X) in avian tissues. J Cell Biol 100:598–605
Horton WA, Machado MM (1988) Extracellular matrix alterations during endochondral ossifications in humans. J Orthop Res 6:793–803
Shapiro IM, Boyde A (1984) Microdissection-elemental analysis of the mineralizing growth cartilage of the normal and rachitic chick. Metab Bone Dis Rel Res 5:317–326
Kakuta S, Golub EE, Haselgrove JC, Chance B, Frasca P, Shapiro IM (1986) Redox studies of the epiphyseal growth cartilage: pyridine nucleotide metabolism and the development of mineralization. J Bone Miner Res 1:433–440
Capasso O, Gionti E, Pontarelli G, Ambesioimpiombato FS, Nitsch L, Tajana G, Cancedda R (1982) The culture of chick embryo chondrocytes and the control of their differentiated functions in vitro. Exp Cell Res 142:197–206
Gerstenfeld LC, Landis WJ (1991) Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system. J Cell Biol 112:501–513
Iwamoto M, Shimazu A, Nakashima K, Suzuki F, Kato Y (1991) Reduction in basic fibroblast growth factor receptor is coupled with terminal differentiation of chondrocytes. J Biol Chem 266:461–467
Kato Y, Iwamoto M, Koike T, Suzuki F, Takano Y (1988) Terminal differentiation and calcification in rabbit chondrocyte cultures grown in centrifuge tubes: regulation by transforming growth factor β and serum factors. Proc Natl Acad Sci USA 85:9552–9556
Kato Y, Shimazu A, Nakashima K, Suzuki F, Jikko A, Iwamoto M (1990) Effects of parathyroid hormone and calcitonin on alkaline phosphatase activity and matrix calcification in rabbit growth plate chondrocyte cultures. Endocrinology 127:114–118
Oettinger HF, Pacifici M (1990) Type X collagen gene expression is transiently up-regulated by retinoic acid treatment in chick chondrocyte cultures. Exp Cell Res 191:292–298
Pacifici M, Golden EB, Iwamoto M, Adams SL (1991) Retinoic acid treatment induces type X collagen gene expression in culture chick chondrocytes. Exp Cell Res 195:38–46
Iwamoto M, Shapiro IM, Yagami K, Boskey AL, Leboy PS, Adams S, Pacifici M (1993) Retinoic acid induces rapid mineralization and expression of mineralization-related genes in chondrocytes. Exp Cell Res 207:413–420
Leboy PS, Vaias L, Uschmann B, Golub E, Adams SL, Pacifici M (1989) Ascorbic acid induces alkaline phosphatase, type X collagen, and calcium deposition in cultured chick chondrocytes. J Biol Chem 264:17281–17286
West DC, Satar A, Kumar S (1985) A simplified in situ solubilization procedure for the determination of DNA and cell number in tissue-cultured mammalian cells. Anal Biochem 147:289–295
Pacifici M, Iozzo RV (1988) Remodelling of the rough endoplasmic reticulum during stimulation of procollagen secretion by ascorbic acid in cultured chondrocytes. J Biol Chem 263:2483–2492
Royce PM, Lowther DA (1979) Fluorimetric determination of DNA in papain digests of cartilage, using ethidium bromide. Connect Tissue Res 6:215–221
Kim Y-J, Sah RL, Doong J-YH, Grodzinsky AJ (1988) Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem 174:168–176
Cheung HS, Ryan LM (1981) A method for determining DNA and chondrocyte content of articular cartilage. Anal Biochem 116:93–97
Fasman GD (1976) Handbook of biochemistry and molecular biology. In: Nucleic acids, vol II, 3rd ed. CRC Press, Boca Raton, pp 300–301
Lowe MG (1989) Glycosyl-phosphatidylinositol: a versatile anchor for cell surface proteins. FASEB J 3:1600–1608
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Teixeira, C.C., Hatori, M., Leboy, P.S. et al. A rapid and ultrasensitive method for measurement of DNA, calcium and protein content, and alkaline phosphatase activity of chondrocyte cultures. Calcif Tissue Int 56, 252–256 (1995). https://doi.org/10.1007/BF00298620
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DOI: https://doi.org/10.1007/BF00298620