Telomere length analysis on cord blood cells by the flow-FISH method

Regéczy, N.; Valent, Sándor; Kormos, Luca; Hajdu, Melinda; Gopcsa, László; Pálóczi, Katalin

doi:10.1163/15685590260461075

Cited by 3 publications

(1 citation statement)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…4G ) in 1.25% C-HS respectively. For comparison, the average telomeric length of cord blood cells was 18.5±3.9%, when compared to 1301 cell line [23] (p<0.05).…”

Section: Resultsmentioning

confidence: 97%

Isolation and Characterization of Human Dental Pulp Derived Stem Cells by Using Media Containing Low Human Serum Percentage as Clinical Grade Substitutes for Bovine Serum

et al. 2012

View full text Add to dashboard Cite

Adult stem cells have been proposed as an alternative to embryonic stem cells to study multilineage differentiation in vitro and to use in therapy. Current culture media for isolation and expansion of adult stem cells require the use of large amounts of animal sera, but animal-derived culture reagents give rise to some questions due to the real possibility of infections and severe immune reactions. For these reasons a clinical grade substitute to animal sera is needed. We tested the isolation, proliferation, morphology, stemness related marker expression, and osteoblastic differentiation potential of Dental Pulp Stem Cells (DPSC) in a chemically defined medium containing a low percentage of human serum, 1.25%, in comparison to a medium containing 10% Fetal Bovine Serum (FBS). DPSCs cultured in presence of our isolation/proliferation medium added with low HS percentage were obtained without immune-selection methods and showed high uniformity in the expression of stem cell markers, proliferated at higher rate, and demonstrated comparable osteoblastic potential with respect to DPSCs cultured in 10% FBS. In this study we demonstrated that a chemically defined medium added with low HS percentage, derived from autologous and heterologous sources, could be a valid substitute to FBS-containing media and should be helpful for adult stem cells clinical application.

show abstract

“…4G ) in 1.25% C-HS respectively. For comparison, the average telomeric length of cord blood cells was 18.5±3.9%, when compared to 1301 cell line [23] (p<0.05).…”

Section: Resultsmentioning

confidence: 97%

Isolation and Characterization of Human Dental Pulp Derived Stem Cells by Using Media Containing Low Human Serum Percentage as Clinical Grade Substitutes for Bovine Serum

et al. 2012

View full text Add to dashboard Cite

show abstract

Automatic telomere length measurements in interphase nuclei by IQ‐FISH

Narath

Lörch²,

Greulich-Bode

et al. 2005

Cytometry Pt A

View full text Add to dashboard Cite

To benefit from the fluorescence-based automatic microscope (FLAME), we have adapted a PNA FISH technique to automatically determine telomere length in interphase nuclei. The method relies on the simultaneous acquisition of pan-telomeric signals and reference probe signals. We compared the quantitative figures to those for existing methods, i.e. Southern blot analysis and quantitative FISH (Q-FISH). Quantitative-FISH on interphase nuclei (IQ-FISH) allows the exact quantification of telomere length in interphase nuclei. Thus, this enables us to obtain not only exact information on the telomere length, but also morphological and topological details. The automatic measurement of large cell numbers allows the measurement of statistically relevant cell populations. Key terms: automatic; quantification; telomere; FISH; microscopy; fluorescence; ALT; senescence Monitoring of telomere length regulation has become an important aspect of stem-cell research, studies on cellular senescence, and cancer research. A number of techniques have been developed to study telomere length in a quantitative manner. The standard procedure is Southern blot hybridization (1). The quantitative fluorescence in situ hybridization (Q-FISH) on metaphase chromosomes allows the quantification of the telomeric FISH signals on individual chromosomes (2-4). Since fluorescence intensity of the telomeric signals was found to be proportional to the size of telomeric repeats, Q-FISH is now widely used (2). For measuring telomere length in interphase nuclei, flow cytometry or fluorescence microscopy can be applied (5-7). Nonadhesive hematopoietic cells seem to be better suited for flow cytometric analyses than solid tumor cells (8,9). Therefore, for the analysis of nonhematopoietic cells, interphase FISH is the method of choice. However, the fluorescence microscopical method to quantify telomere length in interphase nuclei has so far only been performed manually on a restricted number of cells (6,7). To combine the positive aspects of flow cytometric measurements with the ability to quantify individual nuclei by fluorescence microscopical examination, we took advantage of fluorescence-based automatic microscope (FLAME). Thus, all the advantages of interphase measurements, i.e., the analysis of individual cells and the applicability to nonproliferating cells, can be combined with the analyses of statistically relevant cell populations. To develop a reliable method to automatically quantify telomere length in interphase nuclei, we applied a two-color hybridization assay and measured the fluorescence signals with FLAME. We then described the parameters essential for intra-and interexperimental comparisons. We performed intensity measurements in interphase nuclei and compared the results of single channel measurements of the target probe with the results obtained after introducing an internal reference and performing double channel measurements. We validated the quantitative-FISH on interphase nuclei (IQ-FISH) method by measuring telomere lengths of differe...

show abstract