NaCl-aided Hoechst 33258 staining method for DNA quantification and its application
✍ Scribed by A. Yamamoto; T. Araki; K. Fujimori; M. Yamada; H. Yamaguchi; K. Izumi; K. Matsumoto
- Publisher
- Springer
- Year
- 1989
- Tongue
- English
- Weight
- 433 KB
- Volume
- 92
- Category
- Article
- ISSN
- 1432-119X
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✦ Synopsis
We investigated the effect of salt on the fluorescence staining procedure for quantification of the amount of D N A in cell nuclei in situ. F o r this, NaC1 was added at various concentrations to the Hoechst 33258 fluorochrome (Hoe) medium for staining D N A . The fluorescence intensity of free D N A -H o e solution was not changed by the addition of NaC1, but that of the nuclei-Hoe complex in situ increased 4-fold on increasing the NaC1 concentration up to 1 M. SDS polyacrylamide gel electrophoresis showed that histones H I , H2A, and H2B dissociated from cell nuclei in the presence of 1 M NaC1, resulting in increasing accessibility of D N A to the fluorochrome.
The applicability of the NaCl-aided fluorescence staining method was evaluated by measuring the ploidy classes of various cells. The amount of D N A in spermatozoa is half that in 2n hepatocytes, but by the conventional Hoe staining procedure the fluorescence intensity of spermatozoa is higher than that of 2n hepatocytes, due to differences in accessibility of the dye to D N A . In contrast, by the NaC1aided procedure, the fluorescence intensity of 2n hepatocytes was twice that of spermatozoa. The effectiveness of the NaCl-aided Hoe staining method was checked using cultivated human gingival cells and hepatocytes of LEC rats with hereditary hepatitis. In all cases, reasonable proportionality between the fluorescence intensity and the amount of D N A was observed.