NaCl-aided Hoechst 33258 staining method for DNA quantification and its application.
We investigated the effect of salt on the fluorescence staining procedure for quantification of the amount of DNA in cell nuclei in situ. For this, NaCl was added at various concentrations to the Hoechst 33258 fluorochrome (Hoe) medium for staining DNA. The fluorescence intensity of free DNA-Hoe solution was not changed by the addition of NaCl, but that of the nuclei-Hoe complex in situ increased 4-fold on increasing the NaCl concentration up to 1 M. SDS polyacrylamide gel electrophoresis showed that histones H1, H2A, and H2B dissociated from cell nuclei in the presence of 1 M NaCl, resulting in increasing accessibility of DNA 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 DNA in spermatozoa is half that in 2 n hepatocytes, but by the conventional Hoe staining procedure the fluorescence intensity of spermatozoa is higher than that of 2 n hepatocytes, due to differences in accessibility of the dye to DNA. In contrast, by the NaCl-aided procedure, the fluorescence intensity of 2 n 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 DNA was observed.
Yamamoto
A
A
Department of Anatomy, University of Tokushima, Japan.
Araki
T
T
Fujimori
K
K
Yamada
M
M
Yamaguchi
H
H
Izumi
K
K
Matsumoto
K
K
D000818:Animals; D001562:Benzimidazoles; D006690:Bisbenzimidazole; D002467:Cell Nucleus / Q000032:analysis*; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis*; D005470:Fluorometry; D005881:Gingiva / Q000032:analysis / Q000166:cytology; D006801:Humans; D008099:Liver / Q000032:analysis / Q000166:cytology; D008297:Male; D051381:Rats; D012965:Sodium Chloride / Q000032:analysis; D013094:Spermatozoa / Q000032:analysis / Q000166:cytology; D013552:Swine
D001562:Benzimidazoles; D012965:Sodium Chloride; D004247:DNA; D006690:Bisbenzimidazole