Significantly improved precision of cell migration analysis in time-lapse video microscopy through use of a fully automated tracking system

Huth, Johannes; Buchholz, Malte; Kraus, Johann M.; Schmücker, Martin; Wichert, Goetz von; Krndija, Denis; Seufferlein, Thomas; Gress, Thomas M.; Kestler, Hans A.

doi:10.1186/1471-2121-11-24

Cited by 85 publications

(67 citation statements)

References 44 publications

(53 reference statements)

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“…Migration was measured by counting ten fields of view under a microscope covering about 70-80% of the insert area of the fixed and stained cells (0.2% crystal violet) migrating towards the conditioned media and normalised to the number of cells that migrated towards mock conditioned media. The number of migrated cells was also validated using the via TimeLapseAnalyzer program (Huth et al 2010).…”

Section: Cell Migration Experimentsmentioning

confidence: 99%

“…After incubation for 24 h, at 37 8C, the wells were photographed at a fourfold magnification using a Canon 450 EOS camera connected to the microscope (Olympus IMT2). The length and junctions of the generated tubes were analysed using the TimeLapseAnalyzer program (Huth et al 2010). Each experiment was carried out at least in triplicate with independently obtained conditioned media.…”

Section: Tube Formation Assaysmentioning

confidence: 99%

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CUX1: a modulator of tumour aggressiveness in pancreatic neuroendocrine neoplasms

Krug¹,

Kühnemuth²,

Griesmann³

et al. 2014

Endocrine-Related Cancer

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Pancreatic neuroendocrine neoplasms (PNENs) constitute a rare tumour entity, and prognosis and treatment options depend on tumour-mediating hallmarks such as angiogenesis, proliferation rate and resistance to apoptosis. The molecular pathways that determine the malignant phenotype are still insufficiently understood and this has limited the use of effective combination therapies in the past. In this study, we aimed to characterise the effect of the oncogenic transcription factor Cut homeobox 1 (CUX1) on proliferation, resistance to apoptosis and angiogenesis in murine and human PNENs. The expression and function of CUX1 were analysed using knockdown and overexpression strategies in Ins-1 and Bon-1 cells, xenograft models and a genetically engineered mouse model of insulinoma (RIP1Tag2). Regulation of angiogenesis was assessed using RNA profiling and functional tube-formation assays in HMEC-1 cells. Finally, CUX1 expression was assessed in a tissue microarray of 59 human insulinomas and correlated with clinicopathological data. CUX1 expression was upregulated during tumour progression in a time-and stage-dependent manner in the RIP1Tag2 model, and associated with pro-invasive and metastatic features of human insulinomas. Endogenous and recombinant CUX1 expression increased tumour cell proliferation, tumour growth, resistance to apoptosis, and angiogenesis in vitro and in vivo. Mechanistically, the pro-angiogenic effect of CUX1 was mediated via upregulation of effectors such as HIF1a and MMP9. CUX1 mediates an invasive pro-angiogenic phenotype and is associated with malignant behaviour in human insulinomas. Key Words" CUX1 " pancreatic neuroendocrine neoplasms " angiogenesis " RIP1Tag2" insulinomas

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Section: Cell Migration Experimentsmentioning

confidence: 99%

Section: Tube Formation Assaysmentioning

confidence: 99%

CUX1: a modulator of tumour aggressiveness in pancreatic neuroendocrine neoplasms

Krug¹,

Kühnemuth²,

Griesmann³

et al. 2014

Endocrine-Related Cancer

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“…Manual image analysis is labor intensive, slow and carries the risk of human errors and bias (Huth et al, 2010). However, most probably the greatest shortfall of the human eye is that it is not quantitative and cannot always distinguish subtle effects.…”

Section: Manual Image Analysismentioning

confidence: 99%

Getting the whole picture: combining throughput with content in microscopy

Rimon

Schuldiner

2011

Journal of Cell Science

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Commentary 3743Introduction Technological developments in the past years have led to the emergence of high-throughput methods in cell biology (Box 1), where multiple perturbations of a system are automatically repeated in a controlled and identical fashion. These capabilities allow the acquisition of vast amounts of data on a biological system. Evaluating all aspects of such data using computational and mathematical tools can then lead to an unbiased systematic representation of the process studied (Ahn et al., 2006). Technical breakthroughs, often driven by the pharmaceutical industry, have pushed this field forwards and have led to major advances in drug screening (reviewed by Zanella et al., 2010) and numerous discoveries in basic biology.The need for systematic and unbiased approaches has always been at the core of scientific efforts. However, the technology that is required for such efforts often displays an inverse relationship between throughput (speed) and content (biological information). Sydney Brenner referred to this phenomenon by saying that highthroughput experiments are in danger of creating "low-input, high-throughput, no-output biology" (Brenner, 2008). Therefore, it remains a major goal to develop high-throughput science that will give all the advantages of being systematic, accurate, fast and unbiased without giving up the requirement to provide profound and highly informative data.Among the variety of high-throughput approaches available to date (Box 1), one of the methods that holds the promise to bridge the gap between these expectations is high-throughput microscopy, often also referred to as high-content screening (HCS). This is mainly owing to the ability of microscopy methods to focus on single cells at a subcellular resolution, in a time-dependent manner and to measure a large number of parameters in each frame. In this Commentary, we focus on the current frontiers of HCS by presenting the wide range of tools that are utilized and the biological questions that can be tackled using such approaches. We also discuss possible ways to increase content while maintaining throughput at all levels of the screen -from sample design and preparation through to the acquisition and analysis capabilities of the high-content imager system. The power of high-throughput microscopyHigh-throughput microscopy can be employed to address a wide spectrum of biological questions. At the basis of this capability lies the ever-growing variety of labeling methods (discussed below) that allow visualization of cellular architecture and function, as well as developmental or behavioral processes (Fig. 1). In addition, the technological advance of biological tools and microscopic platforms now enables screens to be performed in an array of genetic backgrounds, under different growth conditions and at various time points, as well as allowing the comparison of multiple tissues, cell lines or organisms. Combining the richness of visualization approaches with various experimental strategies creates nearly endless options ...

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“…The videos were recorded with a temporal resolution of 5 s using a Hamamatsu ORCA-R2 charge-coupled device (CCD) camera and Cell-R software (Hamamatsu Photonics Deutschland GmbH, Herrsching am Ammersee, Germany). Cell migration rates were determined using Time-Lapse Analyzer software (22). Tracks of the cells that remained rounded after stimulation were manually excluded from the analysis.…”

Section: Time-lapse Recording and Analysis Of Cell Migrationmentioning

confidence: 99%

Acute-Phase Protein α1-Antitrypsin Inhibits Neutrophil Calpain I and Induces Random Migration

Al-Omari

Korenbaum

Ballmaier

et al. 2011

Mol Med

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A rapid recruitment of neutrophils to sites of injury or infection is a hallmark of the inflammatory response and is required for effective host defense against pathogenic stimuli. However, neutrophil-mediated inflammation can also lead to chronic tissue destruction; therefore, a better understanding of the mechanisms underlying neutrophil influx and activation is of critical importance. We have previously shown that the acute phase protein α1-antitrypsin (AAT) inhibits neutrophil chemotaxis. In this study, we examine mechanisms related to the effect of AAT on neutrophil responses. We report a previously unknown function of AAT to inactivate calpain I (μ-calpain) and to induce a rapid cell polarization and random migration. These effects of AAT coincided with a transient rise in intracellular calcium, increase in intracellular lipids, activation of the Rho GTPases, Rac1 and Cdc42, and extracellular signal-regulated kinase (ERK1/2). Furthermore, AAT caused a significant inhibition of nonstimulated as well as formyl-metleu-phe (fMLP)-stimulated neutrophil adhesion to fibronectin, strongly inhibited lipopolysaccharide-induced IL-8 release and slightly delayed neutrophil apoptosis. The results presented here broaden our understanding of the regulation of calpain-related neutrophil functional activities, and provide the impetus for new studies to define the role of AAT and other acute phase proteins in health and disease.

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Significantly improved precision of cell migration analysis in time-lapse video microscopy through use of a fully automated tracking system

Cited by 85 publications

References 44 publications

CUX1: a modulator of tumour aggressiveness in pancreatic neuroendocrine neoplasms

CUX1: a modulator of tumour aggressiveness in pancreatic neuroendocrine neoplasms

Getting the whole picture: combining throughput with content in microscopy

Acute-Phase Protein α1-Antitrypsin Inhibits Neutrophil Calpain I and Induces Random Migration

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