Showing papers by "Teresa S. Hawley published in 2005"

DPSS yellow-green 561-nm lasers for improved fluorochrome detection by flow cytometry†

Abstract: Introduction Blue-green 488-nm laser sources are widespread in flow cytometry but suffer some drawbacks for cell analysis, including their excitation of endogenous proteins (resulting in high cellular autofluorescence) and their less-than-optimal coincidence with the excitation maxima of commonly used fluorochromes, including the phycoerythrins (PE). Longer wavelength lasers such as green helium–neons and, more recently, diode-pumped solid state (DPSS) 532-nm sources have previously been employed to overcome these difficulties and improve overall sensitivity for PE. In this study, we evaluate an even longer wavelength DPSS 561-nm for its ability to improve PE and DsRed fluorescent protein detection sensitivity. Methods A DPSS 561-nm laser emitting at 10 mW was mounted onto a BD LSR II. Mouse thymoma cells labeled with cell surface marker antibodies conjugated to the R- and B-forms of PE were analyzed and compared with conventional 488-nm excitation using the same bandpass filters and signal travel distances. A similar analysis was carried out with cell lines expressing the red fluorescent protein DsRed, several green-yellow excited low molecular weight fluorochromes, and a rhodamine-based caspase substrate. Additionally, cells labeled with PE and co-labeled with fluorescein or simultaneously expressing green fluorescent protein (GFP) were analyzed to determine if PE excitation at 561 nm with simultaneous fluorescein/GFP detection was feasible. Results The DPSS 561-nm laser gave a several-fold improvement in the fluorochrome to autofluorescence ratios between PE-labeled cells and unlabeled controls. Analysis of cells expressing the fluorescent protein DsRed with the DPSS 561-nm source gave a 6–7-fold improvement in sensitivity over 488-nm excitation, and gave excellent excitation of yellow-green excited fluorochromes and rhodamine-based physiological probes. Yellow-green laser light also caused virtually no impingement on the spatially separated fluorescein/GFP detector, a significant problem with green laser sources, and also allowed simultaneous analysis of GFP and PE with virtually no signal overlap or requirement for color compensation. Conclusions DPSS 561-nm laser excitation gave significantly improved sensitivity for both PE-labeled and DsRed expressing cells, with little contamination of a typical fluorescein/GFP detector. Published 2005 Wiley-Liss, Inc.

Bypass of senescence, immortalization, and transformation of human hematopoietic progenitor cells

Abstract: We attempted to extend the lifespan of CD34+ stem/progenitor cells in human cord blood (CB) by transduction with lentiviral vectors carrying the human telomerase catalytic subunit (hTERT) and/or the human papillomavirus type 16 (HPV16) E6 and E7 oncogenes. We found that hTERT was incapable of prolonging the replicative capacity of CB cells maintained under serum-free conditions in the presence of stem cell factor, Flt3 ligand, thrombopoietin, and interleukin-3 beyond 4 months (n=3). However, transduced CB cells cultured in the same cytokine cocktail constitutively expressing HPV16 E6/E7 alone (n=2) or in concert with hTERT (n=9) continued to proliferate, giving rise to permanent (>2 years) cell lines with a CD45+ CD34- CD133+/- CD44+ CD235a+ CD71+ CD203+ CD33+ CD13+ myeloerythroid/mast cell progenitor phenotype. Notably, CB cell cultures expressing only HPV16 E6/E7 went through a crisis period, and the resulting oligoclonal cell lines were highly aneuploid. By comparison, the CB cell lines obtained by coexpression of HPV16 E6/E7 plus hTERT exhibited near-diploid karyotypes with minimal chromosomal aberrations, concomitant with stabilization of telomere length, yet were clonally derived. The immortalized E6/E7 plus hTERT-expressing CB cells were not tumorigenic when injected intravenously or subcutaneously into sublethally irradiated immunodeficient nonobese diabetic/severe combined immunodeficient mice but could be converted to a malignant state by ectopic expression of a v-H-ras or BCR-ABL oncogene. These findings provide new insights into the mechanisms governing the senescence checkpoint of primitive human hematopoietic precursors and establish a paradigm for studies of the multistep process of human leukemogenesis.