Fluorometric assay of DNA in cartilage explants using Hoechst 33258.
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.
Kim
Y J
YJ
Continuum Electromechanics Group, Department of Electrical Engineering and Computer Science, Massachusetts.
Sah
R L
RL
Doong
J Y
JY
Grodzinsky
A J
AJ
D000818:Animals; D001562:Benzimidazoles; D006690:Bisbenzimidazole; D002358:Cartilage, Articular / Q000032:analysis*; D002417:Cattle; D004247:DNA / Q000032:analysis*; D003851:Deoxyribonucleases; D005615:Freezing; D010206:Papain; D012260:Ribonucleases; D013050:Spectrometry, Fluorescence / Q000379:methods*
D001562:Benzimidazoles; D004247:DNA; D003851:Deoxyribonucleases; D012260:Ribonucleases; D010206:Papain; D006690:Bisbenzimidazole