scholarly journals Enhanced γ-H2AX DNA damage foci detection using multimagnification and extended depth of field in imaging flow cytometry

2015 ◽  
Vol 87 (8) ◽  
pp. 717-723 ◽  
Author(s):  
Christopher N. Parris ◽  
Sheba Adam Zahir ◽  
Hussein Al-Ali ◽  
Emma C. Bourton ◽  
Christina Plowman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dna Damage ◽  
Depth Of Field ◽  
Imaging Flow Cytometry ◽  
Extended Depth Of Field

scholarly journals Imaging flow cytometry as a sensitive tool to detect low-dose-induced DNA damage by analyzing 53BP1 and γH2AX foci in human lymphocytes

2015 ◽  
Vol 87 (12) ◽  
pp. 1070-1078 ◽  
Author(s):  
Matus Durdik ◽  
Pavol Kosik ◽  
Jan Gursky ◽  
Lenka Vokalova ◽  
Eva Markova ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dna Damage ◽  
Low Dose ◽  
Human Lymphocytes ◽  
Imaging Flow Cytometry ◽  
Γh2ax Foci ◽  
Sensitive Tool

scholarly journals Development of an In Vitro whole cell Micronucleus Multiplex expansion assay: Using DRAQ5™ and the DNA damage biomarkers ɣH2AX, P53, pH3 along with cell cycle relationships and the MN genotoxic endpoint to assess chemical Mode of Action on an imaging flow cytometry platform with template development

2021 ◽  
Author(s):  
◽  
Danielle S.G. Harte
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Dna Damage ◽  
Mode Of Action ◽  
In Vitro Tests ◽  
Genetic Toxicology ◽  
Whole Cell ◽  
Imaging Flow Cytometry ◽  
Multiplex System

Genetic toxicity testing is the assessment of compounds, and their respective metabolites, potential to cause DNA damage either directly or indirectly. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development to help identify promising drugs that have a low risk potential of causing damage leading to cancer in humans. Despite this, in vitro tests generate a high number of miss-leading positives, the consequences of which lead to unnecessary animal testing and abandoning promising drug candidates. Understanding chemical Mode of Action (MoA) is vital in identifying true genotoxic potential of substances. Here I demonstrate a simple robust protocol for optimised staining of fixed human lymphoblast P53 proficient TK6 cells with the antibodies; Anti-ɣH2AX, Anti-P53 and Ant-pH3S28 along with DRAQ5™ DNA staining in a whole cell multiplex system that is suitable for analysis via microscopy or imaging flow cytometry. Use of the Amnis FlowSight® and ImageStream X Mark II® platform provided a high content high throughput acquisition platform with the sensitivity of flow cytometry and accuracy of image analysis. Using both optimal and suboptimal lasers for fluorophore excitation demonstrated that a multiplex system for DNA damage assessment including MN was possible in un-lysed cells. IDEAS 6.2 template generation allowed for batch processing of data samples extracting the following metrics: Cell Cycle, Micronucleus (MN), ɣH2AX, P53, PH3, G1 ɣH2AX, G1 P53, S ɣH2AX, S P53, G2 ɣH2AX, G2/M P53, Prophase, Metaphase, Anaphase and Abnormal mitosis. Furthermore, high content nature of the platform using imagery allows for identification of rare cellular event assessment particularly preliminary data on biomarker signal found within MN. The system found differences in the biomarker metric responses between the chemicals; MMS, Carbendazim, ARA-C, Vinblastine, Etoposide and Crizotinib suggesting potential for chemical MoA elucidation.

A Novel Camptothecin Derivative 3j Inhibits Nsclc Proliferation Via Induction of Cell Cycle Arrest By Topo I-Mediated DNA Damage

2019 ◽  
Vol 19 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Yang Liu ◽  
Jingyin Zhang ◽  
Shuyun Feng ◽  
Tingli Zhao ◽  
Zhengzheng Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Dna Damage ◽  
Cyclin D ◽  
Dna Relaxation ◽  
Cycle Arrest ◽  
H460 Cell ◽  
H1975 Cell

Objective: The aim of this study is to investigate the inhibitory effect of camptothecin derivative 3j on Non-Small Cell Lung Cancer (NSCLCs) cells and the potential anti-tumor mechanisms. Background: Camptothecin compounds are considered as the third largest natural drugs which are widely investigated in the world and they suffered restriction because of serious toxicity, such as hemorrhagic cystitis and bone marrow suppression. Methods: Using cell proliferation assay and S180 tumor mice model, a series of 20(S)-O-substituted benzoyl 7- ethylcamptothecin compounds were screened and evaluated the antitumor activities in vitro and in vivo. Camptothecin derivative 3j was selected for further study using flow cytometry in NSCLCs cells. Cell cycle related protein cyclin A2, CDK2, cyclin D and cyclin E were detected by Western Blot. Then, computer molecular docking was used to confirm the interaction between 3j and Topo I. Also, DNA relaxation assay and alkaline comet assay were used to investigate the mechanism of 3j on DNA damage. Results: Our results demonstrated that camptothecin derivative 3j showed a greater antitumor effect in eleven 20(S)-O-substituted benzoyl 7-ethylcamptothecin compounds in vitro and in vivo. The IC50 of 3j was 1.54± 0.41 µM lower than irinotecan with an IC50 of 13.86±0.80 µM in NCI-H460 cell, which was reduced by 8 fold. In NCI-H1975 cell, the IC50 of 3j was 1.87±0.23 µM lower than irinotecan (IC50±SD, 5.35±0.38 µM), dropped by 1.8 fold. Flow cytometry analysis revealed that 3j induced significant accumulation in a dose-dependent manner. After 24h of 3j (10 µM) treatment, the percentage of NCI-H460 cell in S-phase significantly increased (to 93.54 ± 4.4%) compared with control cells (31.67 ± 3.4%). Similarly, the percentage of NCI-H1975 cell in Sphase significantly increased (to 83.99 ± 2.4%) compared with control cells (34.45 ± 3.9%) after treatment with 10µM of 3j. Moreover, increased levels of cyclin A2, CDK2, and decreased levels of cyclin D, cyclin E further confirmed that cell cycle arrest was induced by 3j. Furthermore, molecular docking studies suggested that 3j interacted with Topo I-DNA and DNA-relaxation assay simultaneously confirmed that 3j suppressed the activity of Topo I. Research on the mechanism showed that 3j exhibited anti-tumour activity via activating the DNA damage response pathway and suppressing the repair pathway in NSCLC cells. Conclusion: Novel camptothecin derivative 3j has been demonstrated as a promising antitumor agent and remains to be assessed in further studies.

scholarly journals Semi-automated classification of colonial Microcystis by FlowCAM imaging flow cytometry in mesocosm experiment reveals high heterogeneity during seasonal bloom

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yersultan Mirasbekov ◽  
Adina Zhumakhanova ◽  
Almira Zhantuyakova ◽  
Kuanysh Sarkytbayev ◽  
Dmitry V. Malashenkov ◽  
...  
Keyword(s):  
Machine Learning ◽  
Flow Cytometry ◽  
Spatial Resolution ◽  
Mesocosm Experiment ◽  
Imaging Flow Cytometry ◽  
Leibler Divergence ◽  
Temporal And Spatial ◽  
High Level ◽  
Training Sets

AbstractA machine learning approach was employed to detect and quantify Microcystis colonial morphospecies using FlowCAM-based imaging flow cytometry. The system was trained and tested using samples from a long-term mesocosm experiment (LMWE, Central Jutland, Denmark). The statistical validation of the classification approaches was performed using Hellinger distances, Bray–Curtis dissimilarity, and Kullback–Leibler divergence. The semi-automatic classification based on well-balanced training sets from Microcystis seasonal bloom provided a high level of intergeneric accuracy (96–100%) but relatively low intrageneric accuracy (67–78%). Our results provide a proof-of-concept of how machine learning approaches can be applied to analyze the colonial microalgae. This approach allowed to evaluate Microcystis seasonal bloom in individual mesocosms with high level of temporal and spatial resolution. The observation that some Microcystis morphotypes completely disappeared and re-appeared along the mesocosm experiment timeline supports the hypothesis of the main transition pathways of colonial Microcystis morphoforms. We demonstrated that significant changes in the training sets with colonial images required for accurate classification of Microcystis spp. from time points differed by only two weeks due to Microcystis high phenotypic heterogeneity during the bloom. We conclude that automatic methods not only allow a performance level of human taxonomist, and thus be a valuable time-saving tool in the routine-like identification of colonial phytoplankton taxa, but also can be applied to increase temporal and spatial resolution of the study.

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