FLUOROMETRIC ASSAY OF DNA IN CARTILAGE EXPLANTS USING HOECHST-33258
FLUOROMETRIC ASSAY OF DNA IN CARTILAGE EXPLANTS USING HOECHST-33258
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DOI：
10.1016/0003-2697(88)90532-5
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发表时间：
1988-10-01
影响因子：
2.9
通讯作者：
GRODZINSKY, AJ
中科院分区：
文献类型：
--
作者：
KIM, YJ;SAH, RLY;GRODZINSKY, AJ
一个简单的两步荧光测定DNA软骨外植体，利用双苯并咪唑染料Hoechst 33258，描述。用木瓜蛋白酶消化制备软骨外植体进行实验。等分消化液与染料溶液混合，测量荧光发射。染料的荧光增强对DNA具有特异性，对DNase的敏感性为97%，对RNase具有抗性。此外，非DNA组织成分引起的干扰很少或没有，因为DNA引起的荧光增强与木瓜蛋白酶消化软骨的存在无关。通过对分离的软骨细胞进行测定，计算出每个软骨细胞的DNA含量为7.7 pg。该试验稳定至少2小时，对低至6 ng的DNA或相当于少于1000个细胞敏感。这种方法比其他已建立的软骨DNA测定法有优势，在软骨外植体的代谢研究中可能特别有用。
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.