High-Throughput Detection of DNA Double-Strand Breaks Using Image Cytometry

Fowler, Tyler L.; Bailey, Alison M.; Bednarz, Bryan; Kimple, Randall J.

doi:10.2144/000114248

Cited by 11 publications

(13 citation statements)

References 4 publications

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“…A novel high-throughput irradiator utilizing a 50 kVp x-ray beam spectrum was used to deliver 4 gray (Gy) as previously described (33, 34). Cells were fixed in 4% paraformaldehyde 4 hours later, permeabilized in 90% methanol, blocked, and incubated with γ-H2AX primary antibody (1:500) overnight.…”

Section: Methodsmentioning

confidence: 99%

AXL Is a Logical Molecular Target in Head and Neck Squamous Cell Carcinoma

Brand

Iida

Stein

et al. 2015

Clinical Cancer Research

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101

103

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Purpose Head and neck squamous cell carcinoma (HNSCC) represents the eighth most common malignancy worldwide. Standard of care treatments for HNSCC patients include surgery, radiation and chemotherapy. Additionally, the anti-epidermal growth factor receptor (EGFR) monoclonal antibody cetuximab is often used in combination with these treatment modalities. Despite clinical success with these therapeutics, HNSCC remains a difficult to treat malignancy. Thus, identification of new molecular targets is critical. Experimental Design In the current study, the receptor tyrosine kinase AXL was investigated as a molecular target in HNSCC using established cell lines, HNSCC patient derived xenografts (PDXs), and human tumors. HNSCC dependency on AXL was evaluated with both anti-AXL siRNAs and the small molecule AXL inhibitor R428. Furthermore, AXL inhibition was evaluated with standard of care treatment regimes used in HNSCC. Results AXL was found to be highly overexpressed in several models of HNSCC, where AXL was significantly associated with higher pathologic grade, presence of distant metastases and shorter relapse free survival in patients with HNSCC. Further investigations indicated that HNSCC cells were reliant on AXL for cellular proliferation, migration, and invasion. Additionally, targeting AXL increased HNSCC cell line sensitivity to chemotherapy, cetuximab, and radiation. Moreover, radiation resistant HNSCC cell line xenografts and PDXs expressed elevated levels of both total and activated AXL, indicating a role for AXL in radiation resistance. Conclusion Collectively, this study provides evidence for the role of AXL in HNSCC pathogenesis and supports further pre-clinical and clinical evaluation of anti-AXL therapeutics for the treatment of patients with HNSCC.

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Section: Methodsmentioning

confidence: 99%

AXL Is a Logical Molecular Target in Head and Neck Squamous Cell Carcinoma

Brand

Iida

Stein

et al. 2015

Clinical Cancer Research

Self Cite

101

103

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“…The monochromator setting was used to measure excitation/emission wavelengths of 500/526 (green) to assess DNA intensity and 460/650 (red) to assess autophagy (10). The MiniMax setting was used to count the number of cells per well as we have previously described (11). The red intensity signal per well was divided by the green intensity or the cell count to assess the level of autophagy and to assess the efficacy of the two normalization methods: controlling to green intensity as recommended by Thome et al vs.…”

Section: Table 1 Comparison Of Commonly Used Methods For Studying Aumentioning

confidence: 99%

High-throughput quantitative detection of basal autophagy and autophagic flux using image cytometry

SenthilKumar¹,

Skiba

Kimple

2019

Preprint

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Quantitative assessment of changes in macro-autophagy is often performed through manual quantification of the number of LC3B foci in immunofluorescence microscopy images. This method is highly laborious, subject to image-field selection and foci-counting bias, and is not sensitive for analyzing changes in basal autophagy. Alternative methods such as flow cytometry and transmission electron microscopy require highly specialized, expensive instruments and time-consuming sample preparation. Immunoblots are prone to exposure-related variations and noise that prevent accurate quantification. We present herein a high-throughput, inexpensive, reliable, and objective method for studying basal level and flux changes in late-stage autophagy using image cytometry and acridine orange staining.

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“…Quantitative luminescence measurements of biomolecules, single cells and tissue specimens in solid phase are particularly valuable for identification and unambiguous confirmation of rare cell types [1][2][3] , time-lapse study of live cells [4][5][6] , profiling of subcellular components and biomolecular expressions [7][8][9] , and a broad range of other diagnostics applications [10][11][12] . The existing techniques based on digital microscopy [13][14][15][16] , however, are time-consuming and resource-demanding, as images are typically captured for the entire sample area, or even through three-dimensional space [17][18][19][20] , followed by stitching and processing to identify and quantitate targets of interest.…”

Section: Introductionmentioning

confidence: 99%

High-Precision Pinpointing of Luminescent Targets in Encoder-Assisted Scanning Microscopy Allowing High-Speed Quantitative Analysis

Zheng

Zhao

et al. 2015

Anal. Chem.

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The new agreement specifically addresses what authors can do with different versions of their manuscript -e.g. use in theses and collections, teaching and training, conference presentations, sharing with colleagues, and posting on websites and repositories. The terms under which these uses can occur are clearly identified to prevent misunderstandings that could jeopardize final publication of a manuscript (Section II, Permitted Uses by Authors). Easy Reference User Guide Posting Submitted Works on Websites and Repositories:A digital file of the unedited manuscript version of a Submitted Work may be made publicly available on websites or repositories (e.g. the Author's personal website, preprint servers, university networks or primary employer's institutional websites, third party institutional or subject-based repositories, and conference websites that feature presentations by the Author(s) based on the Submitted Work) under the following conditions:• The posting must be for non-commercial purposes and not violate the ACS' "Ethical Guidelines to Publication of Chemical Research" (see http://pubs.acs.org/ethics).• If the Submitted Work is accepted for publication in an ACS journal, then the following notice should be included at the time of posting, or the posting amended as appropriate: "This document is the unedited Author's version of a Submitted Work that was subsequently accepted for publication in [JournalTitle], copyright © American Chemical Society after peer review. To access the final edited and published work see [insert ACS Articles on Request author-directed link to Published Work, see http://pubs.acs.org/page/policy/articlesonrequest/index.html]." Note: It is the responsibility of the Author(s) to confirm with the appropriate ACS journal editor that the timing of the posting of the Submitted Work does not conflict with journal prior publication/embargo policies (see http://pubs.acs.org/page/policy/prior/index.html)If any prospective posting of the Submitted Work, whether voluntary or mandated by the Author(s)' funding agency, primary employer, or, in the case of Author(s) employed in academia, university administration, would violate any of the above conditions, the Submitted Work may not be posted. In these cases, Author(s) may either sponsor the immediate public availability of the final Published Work through participation in the feebased ACS AuthorChoice program (for information about this program see http://pubs.acs.org/page/policy/authorchoice/index.html) or, if applicable, seek a waiver from the relevant institutional policy. July, 2016 ABSTRACTCompared with routine microscopy imaging of a few analytes at a time, rapid scanning through the whole sample area of a microscope slide to locate every single target object offers many advantages in terms of simplicity, speed, throughput, and potential for robust quantitative analysis. Existing techniques that accommodate solid-phase samples incorporating individual micron-sized targets generally rely on digital microscopy and image analysis, with in...

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High-Throughput Detection of DNA Double-Strand Breaks Using Image Cytometry

Cited by 11 publications

References 4 publications

AXL Is a Logical Molecular Target in Head and Neck Squamous Cell Carcinoma

AXL Is a Logical Molecular Target in Head and Neck Squamous Cell Carcinoma

High-throughput quantitative detection of basal autophagy and autophagic flux using image cytometry

High-Precision Pinpointing of Luminescent Targets in Encoder-Assisted Scanning Microscopy Allowing High-Speed Quantitative Analysis

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