Open AccessBiophysical identification and sorting of high metastatic variants from B16 melanoma tumor
Author(s) -
Lessin Stuart R.,
Abraham Samuel R.,
Nicolini Claudio
Publication year - 1982
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/cyto.990020609
Subject(s) - biology , microbiology and biotechnology , dapi , flow cytometry , chromatin , acridine orange , fluorescamine , ethidium bromide , population , cytometry , cell sorting , cell , staining , cell culture , nuclear dna , dna , fluorescence , biochemistry , genetics , gene , physics , demography , quantum mechanics , sociology , mitochondrial dna
Chromatin structure, in terms of higher order nuclear‐DNA condensation (scanning cytometry) and in terms of acridine orange primary binding sites (flow cytometry), is analyzed and shown to be significantly different between high (B16‐F10) and low (B16‐F1) metastatic variants of B16 melanoma. Furthermore, double staining of B16‐F10 and B16‐F1 with ethidium bromide (chromatin) and fluorescamine (membranes) provides the identification of a homogeneous subpopulation of cells with enhanced metastatic potential based on differential fluorescamine uptake. Fluorescamine uptake and poststaining viability is shown to be dependent upon the dye/cell ratio at which staining occurs. Utilizing a sterile cell sorting technique, a subpopulation of B16‐F10 with increased fluorescamine uptake representing 30% of the total “intact cell” population was isolated by means of a fluorescence activated cell sorter and replated in vitro . This subpopulation when assayed in vivo produced significantly more pulmonary metastases than its parent cell line. Scanning cytometry of the Feulgen stained sorted subpopulation reveals that the cells possess a unique nuclear morphometry characterized by a 2C‐3C DNA content and a large nuclear area (disperse chromatin). Finally, when we assay simultaneously for nuclear‐DNA organization and cell membrane organization a progressive uncoupling between nuclear and cell morphometry is apparent in B16‐F10 (versus B16‐F1).