The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late-apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states.
We investigated the frequencies of early populations of progenitors in aplastic anaemia (AA) bone marrow, from patients with a range of disease severity, compared with normal. Double-colour immunofluorescent staining for CD34 and CD33 was carried out on bone marrow mononuclear cells (BMMC) and analysed using fluorescence activated cell sorting (FACS). AA CD34+ cells were reduced by 68% compared to normal. In addition, AA CD33+ cells and the three progenitor subsets (CD34+/CD33-, CD34+/CD33+ and CD34-/CD33+) were reduced by 44-80%. Our data lend further support for an early stem cell deficiency in AA.
Summary. Osteoclasts form in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of Nfkb ligand (RANKL), a membrane-bound differentiation factor that is now available as a soluble recombinant molecule. Acquisition of the osteoclast phenotype [the a v b 3 subunit of the vitronectin receptor (VNR)-, calcitonin receptor (CTR)-and F-actin ring-positive cells] is associated with loss of monocyte/macrophage-associated integrins, specifically CD11b, CD11c and CD18. We hypothesized that differences in the osteoclast integrin adhesion molecule profile may exist in osteoclasts generated in stromal cell-rich and in stromal-free conditions. Unlike osteoclasts generated in vivo, F-actin ring-positive (resorbing) osteoclasts formed in soluble RANKL in vitro, in the absence of stromal cells, and co-expressed CD11c and CD18. However, when osteoclasts were generated from peripheral blood mononuclear cells (PBMNCs) in co-cultures with the murine bone marrow stromal cell line 218 (which does not produce membranebound RANKL) in the presence of soluble RANKL, CD11c and CD18 were not expressed by osteoclasts. These findings indicate that the persistent expression of CD11c and CD18 is not accounted for by RANKL being presented in a soluble form and that membrane-bound RANKL is not required for the normal integrin expression in resorbing osteoclasts. This study demonstrates that potentially misleading information may arise by using data obtained from osteoclasts generated in the absence of stromal cells as they do not completely reflect the situation in vivo.
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