Correlation between terminal restriction fragments and flow‐<scp>FISH</scp> measures in samples over wide range telomere lengths

Carbonari, Maurizio; Tedesco, Tiziana; Fiorilli, Massimo

doi:10.1111/cpr.12086

Cited by 5 publications

(5 citation statements)

References 17 publications

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“…Both techniques could detect telomeres between 5 and 16 kb, and the correlation measured of 0.41 was similar to previously described measurements by other groups (34). In our study, the assays produced similar results, with Flow-FISH showing consistently higher measurements, but not significantly different.…”

Section: Discussionsupporting

confidence: 87%

Telomere content measurement in human hematopoietic cells: Comparative analysis of qPCR and Flow‐FISH techniques

et al. 2016

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Abnormal telomere lengths have been linked to cancer and other hematologic disorders. Determination of mean telomere content (MTC) is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Here, we compared a quantitative Polymerase Chain Reaction approach (qPCR) and a flow cytometric approach, fluorescence in situ hybridization (Flow-FISH), to evaluate telomere content distribution in total patient peripheral blood mononuclear cells or specific cell populations. Flow-FISH is based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA) probe and DNA staining with propidium iodide. We showed that both qPCR and Flow-FISH provide a robust measurement, with Flow-FISH measuring a relative content longer than qPCR at a single cell approach and that TRF2 fluorescence intensity did not correlate with MTC. Both methods showed comparable telomere content reduction with age, and the rate of relative telomere loss was similar. Published 2016 Wiley Periodicals Inc. This article is a US government work and, as such, is in the public domain in the United States of America.

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Section: Discussionsupporting

confidence: 87%

Telomere content measurement in human hematopoietic cells: Comparative analysis of qPCR and Flow‐FISH techniques

et al. 2016

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“…Flow cytometric analysis was performed to determine telomere length, according to a published fluorescence in situ hybridization protocol 33 (Flow‐FISH).…”

Section: Methodsmentioning

confidence: 99%

The proliferation of belatacept-resistant T cells requires early IFNα pathway activation

Herr

Desterke

Bargiel

et al. 2022

American Journal of Transplantation

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Belatacept was developed to replace calcineurin inhibitors in kidney transplantation. Its use is associated with better kidney transplant function, a lower incidence of anti‐donor antibodies and higher graft survival. However, it is also associated with a higher risk of cellular rejection. We studied the activation and proliferation mechanisms of belatacept‐resistant T lymphocytes (TLs), to identify new pathways for control. We performed a transcriptomic analysis on CD4+CD57+PD1− memory TLs, which are responsible for a higher incidence of graft rejection, after allogeneic stimulation with activated dendritic cells (aDCs) in the presence or absence of belatacept. After six hours of contact with aDCs, the (CD4+CD57+PD1−) (CD4+CD57+PD1+) and (CD4+CD57−) lymphocytes had different transcriptional profiles with or without belatacept. In the CD4+CD57+PD1−population, the IFNα‐dependent activation pathway was positively overrepresented, and IRF7 transcript levels were high. IRF7 was associated with IFNα/β and IL‐6 regulation. The inhibition of both these cytokines in a context of belatacept treatment inhibited the proliferation of CD4+CD57+PD1− T cells. Our results show that IRF7 is rapidly upregulated in belatacept‐resistant CD4+CD57+PD1− TLs. The inhibition of type I IFN or IL‐6 in association with belatacept treatment reduces the proliferation of belatacept‐resistant TLs, paving the way for new treatments for use in organ transplantation.

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“…However, the use of formamide on whole cells in flow‐FISH was only described in 1998 to measure the average length of telomeres . Our laboratory has published some papers on this subject, and in 2014 we introduced a negative control in flow‐FISH consisting of cells treated with a low ionic strength solution containing formamide . We were therefore able to observe that it was possible to treat cells in formamide at room temperature to obtain sufficient stoichiometric DNA staining.…”

Section: Introductionmentioning

confidence: 99%

New use for an old reagent: Cell cycle analysis of DNA content using flow cytometry in formamide treated cells

Carbonari

2016

Cytometry Pt A

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Formamide has long been one of the most widely used reagents in the study of nucleic acids. However, the use of formamide for treating cells to be analyzed by flow cytometry is a recent development and is restricted to measuring telomere lengths by flow-FISH. In this field, we have published several papers in order to observe the effects of formamide treatment on cells at room temperature. We therefore discovered that, with suitable modifications, a short and simple incubation in this ionizing solvent facilitates cell cycle analysis by flow cytometry, equivalent or superior to that obtained with treatments in alcohol, acetone or detergent in hypotonic solution. Even using a bulky and problematic stain (low quantum efficiency and G-C base preference), such as 7-aminoactinomycin D (7-AAD) which, on the other hand, has the advantage of being excited at 488 nm and does not bind to the RNA, it is possible to obtain excellent coefficients of variation and (G2-M) mode/(G0-G1) mode ratios. These parameters, especially if stained cells are washed before acquisition, arrive at optimal values. It is noteworthy that the ability to wash the cells stained for DNA content analysis without affecting the stoichiometry of the staining has not been described elsewhere in the literature. With formamide treatment the doublets are practically absent, sample recovery is efficient, as well as the preservation of physical parameters, and the stained cells can be stored for at least 10 days at room temperature before acquisition. V C 2016 International Society for Advancement of Cytometry

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Correlation between terminal restriction fragments and flow‐FISH measures in samples over wide range telomere lengths

Cited by 5 publications

References 17 publications

Telomere content measurement in human hematopoietic cells: Comparative analysis of qPCR and Flow‐FISH techniques

Telomere content measurement in human hematopoietic cells: Comparative analysis of qPCR and Flow‐FISH techniques

The proliferation of belatacept-resistant T cells requires early IFNα pathway activation

New use for an old reagent: Cell cycle analysis of DNA content using flow cytometry in formamide treated cells

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