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.
- Alkaline phosphatase
- DNA analysis
- Hoechst dye
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism
- Orthopedics and Sports Medicine