PMID: 7538446Mar 1, 1995Paper

A rapid and ultrasensitive method for measurement of DNA, calcium and protein content, and alkaline phosphatase activity of chondrocyte cultures

Calcified Tissue International
Cristina C TeixeiraI M Shapiro

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 con...Continue Reading

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Citations

Sep 1, 1997·Journal of Cellular Biochemistry·P S LeboyR A Venezian
Jun 22, 1999·Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society·A El-GhannamI M Shapiro
Mar 29, 2012·Rheumatology International·Pingping Chen-AnAnne-Christine Bay-Jensen
Apr 23, 2010·Annals of Biomedical Engineering·Jie JiangHelen H Lu
Jan 13, 2001·Archives of Gerontology and Geriatrics·E Bazsó-Dombi Zs -Nagy I
Sep 20, 2008·Chemical Reviews·Adele L Boskey, Rani Roy
Sep 2, 2008·Tissue Engineering. Part a·Serafim M OliveiraCristina C Teixeira
Oct 23, 1998·Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research·S W VolkP S Leboy
Dec 10, 2002·Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research·E A Cavalcanti-AdamC S Adams
Jul 21, 2009·Biomaterials·Isabel F AmaralC J Kirkpatrick
Apr 4, 2003·Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research·Cristina C TeixeiraIrving M Shapiro
Sep 15, 2010·Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society·Glyn D PalmerCristina C Teixeira
Feb 27, 2010·Journal of Biomedical Materials Research. Part a·Serafim M OliveiraCristina C Teixeira
Nov 2, 2005·Biochemical and Biophysical Research Communications·Jie JiangHelen H Lu
Dec 7, 2006·Cellular & Molecular Biology Letters·Michal Opas, Marc P Fadel
Mar 19, 2014·Journal of Biomedical Materials Research. Part a·Monica MontesiAnna Tampieri
Aug 1, 1997·Journal of Biomedical Materials Research·A El-GhannamI M Shapiro
Dec 17, 2015·Journal of Dental Research·M AlikhaniC C Teixeira
Jun 5, 2003·Clinical Orthopaedics and Related Research·Jean C GanIrving M Shapiro
Jun 5, 2003·Clinical Orthopaedics and Related Research·Jean C GanIrving M Shapiro
Jan 14, 2004·Canadian Journal of Physiology and Pharmacology·David CauviOdile M Chabaud
Aug 1, 1996·Journal of Cellular Physiology·R RajpurohitI M Shapiro
Aug 19, 2000·American Journal of Physiology. Endocrinology and Metabolism·D CauviO Chabaud
Aug 14, 2001·American Journal of Physiology. Endocrinology and Metabolism·D CauviO Chabaud
Jan 31, 2002·Journal of Orthopaedic Science : Official Journal of the Japanese Orthopaedic Association·Athanasios V Papavasiliou

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