Kim Y J, Sah R L, Doong J Y, Grodzinsky A J
Continuum Electromechanics Group, Department of Electrical Engineering and Computer Science, Massachusetts.
Anal Biochem. 1988 Oct;174(1):168-76. doi: 10.1016/0003-2697(88)90532-5.
A simple two-step fluorometric assay of DNA in cartilage explants, utilizing the bisbenzimidazole dye Hoechst 33258, is described. Cartilage explants were prepared for assay by digestion with papain. Aliquots of the digest were mixed with dye solution, and the fluorescence emission measured. The enhancement in fluorescence of dye was specific for DNA, as demonstrated by 97% sensitivity to DNase and resistance to RNase. In addition, little or no interference was caused by non-DNA tissue components, since DNA caused an equal enhancement in fluorescence independent of the presence of papain-digested cartilage. By performing the assay on isolated chondrocytes, the cellular content of DNA was computed to be 7.7 pg per chondrocyte. The assay was stable for at least 2 h and sensitive to as little as 6 ng of DNA or equivalently less than 1000 cells. This procedure offers advantages over other established DNA assays of cartilage and may be especially useful in metabolic studies of cartilage explants.
本文描述了一种利用双苯并咪唑染料Hoechst 33258对软骨外植体中的DNA进行简单两步荧光测定的方法。通过木瓜蛋白酶消化制备用于测定的软骨外植体。将消化液的等分试样与染料溶液混合，并测量荧光发射。染料荧光的增强对DNA具有特异性，这通过对DNase的97%敏感性和对RNase的抗性得到证明。此外，非DNA组织成分几乎不产生干扰，因为无论是否存在木瓜蛋白酶消化的软骨，DNA都会使荧光产生同等程度的增强。通过对分离的软骨细胞进行该测定，计算出每个软骨细胞的DNA细胞含量为7.7 pg。该测定至少在2小时内稳定，对低至6 ng的DNA或等效地少于1000个细胞敏感。该方法比其他已确立的软骨DNA测定方法具有优势，可能在软骨外植体的代谢研究中特别有用。
