A RAPID AND ULTRASENSITIVE METHOD FOR MEASUREMENT OF DNA, CALCIUM ANDPROTEIN-CONTENT, AND ALKALINE-PHOSPHATASE ACTIVITY OF CHONDROCYTE CULTURES

Most investigators are cognizant of the problems inherent in counting cells embedded in a complex and abundant extracellular matrix. To over come these obstacles, we developed a new method of isolating nucleic a cids 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. T he cells were treated with triton X-100 and the nucleic acid content o f the extract was determined by measuring DNA fluorescence in the pres ence 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 c ould 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 measurement s 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 c ulture. In addition, as triton X-100 is a nonionic detergent, we were able to measure cell and matrix proteins; moreover, the presence of th e triton maintained the catalytic state of alkaline phosphatase. We co nclude that this procedure provides a simple and rapid approach to mea suring major indicators of chondrocyte maturation and function.