[Inter-organ differences of the cytometric DNA content in mice: relation of the staining method].
With one step DNA staining methods including cell membrane lysis and RNase treatment, we regularly observed a higher fluorescence emission in liver nuclei compared to bone marrow nuclei in C57BL/6 mice. Therefore this study was conducted in order to emphasize such a phenomenon in other organs and to assess if higher fluorescence emission was related to higher DNA content or staining procedure failure. Liver, bone marrow and testis were removed from Swiss, BDF and C57BL/6 mice. The following samples were prepared: 1) liver cells with TRBC (TRBC = Trout Red Blood Cells = internal standards), 2) bone marrow cells with TRBC, 3) testis cells with TRBC and 4) mixtures of liver, bone marrow and testis cells. The staining procedures were: A) one step pH 10 procedure described by Vindelov (Virchows Arch. B. Cell Path., 1977, 24, 227-242), B) same procedure with twice RNase concentration, C) first method with twice NP 40 concentration and D) three steps procedure including Trypsin and Spermine treatment (Vindelov et al., Cytometry, 1983, 3, 323-327). In protocols A, B and C, "Diploid cells/TRBC" ratio differed significantly between liver, bone marrow and testis nuclei. Moreover, 3 distinct populations of diploid cells were present in samples 4. In protocol D, "Diploid cells/TRBC" ratio were identical between liver, bone marrow and testis nuclei. In samples 4, only 1 population of diploid cells has been observed. This study shows that DNA stabilization by polyamine and protein degradation by protease could act on Propidium Iodide fixation and/or fluorescence emission, with significant differences according to the origin of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Vago
P
P
Laboratoire de Biologie Cellulaire et d'Immunogénétique, Faculté de Médecine, Nîmes.
Aboudkhil
S
S
Aguilar
V
V
Bureau
J P
JP
D000818:Animals; D001853:Bone Marrow / Q000032:analysis; D003584:Cytological Techniques; D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods; D008099:Liver / Q000032:analysis; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D013737:Testis / Q000032:analysis
D004247:DNA
Différences inter-organes du contenu cytométrique en ADN chez la Souris: relations avec la méthode de coloration.