A method to estimate cell cycle time and growth fraction using bromodeoxyuridine-flow cytometry data from a single sample

Eidukevičius, Rimantas; Characiejus, Dainius; Janavičius, Ramūnas; Kazlauskaitė, Nijolė; Pašukonienė, Vita; Mauricas, Mykolas; Otter, W. Den

doi:10.1186/1471-2407-5-122

Cited by 23 publications

(21 citation statements)

References 18 publications

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“…In order to determine the time spent in each of the cell cycle and mitotic phases we used the absolute cell numbers obtained after 96 hours of asynchronous culture. The majority of the cells had undergone three rounds of division, which is consistent with the Jurkat IMT of approximately 24 hours (22). We noted that cells from the fourth division were bunched in the early cycle phases while those from round two were grouped in the later phases.…”

Section: Methodssupporting

confidence: 72%

An imaging flow cytometric method for measuring cell division history and molecular symmetry during mitosis

et al. 2011

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Asymmetric cell division is an important mechanism for generating cellular diversity, however, techniques for measuring the distribution of fate-regulating molecules during mitosis have been hampered by a lack of objectivity, quantitation, and statistical robustness. Here we describe a novel imaging flow cytometric approach that is able to report a cells proliferative history and cell cycle position using dye dilution, pH3, and PI staining to then measure the spatial distribution of fluorescent signals during mitosis using CCD-derived imagery. Using Jurkat cells, resolution of the fluorescently labeled populations was comparable to traditional PMT based cytometers thus eliminating the need to sort cells with specific division histories for microscopy. Subdividing mitotic stages by morphology allowed us to determine the time spent in each cell cycle phase using mathematical modeling approaches. Furthermore high sample throughput allowed us to collect statistically relevant numbers of cells without the need to use blocking agents that artificially enrich for mitotic events. The fluorescent imagery was used to measure PKCf protein and EEA-11 endosome distribution during different mitotic phases in Jurkat cells. While telophase cells represented the favorable population for measuring asymmetry, asynchronously dividing cells spent approximately 43 seconds in this stage, explaining why they were present at such low frequencies. This necessitated the acquisition of large cell numbers. Interestingly we found that PKCf was inherited asymmetrically in 2.5% of all telophasic events whereas endosome inheritance was significantly more symmetrical. Furthermore, molecular polarity at early mitotic phases was a poor indicator of asymmetry during telophase highlighting that, though rare, telophasic events represented the best candidates for asymmetry studies. In summary, this technique combines the spatial information afforded by fluorescence microscopy with the statistical wealth and objectivity of traditional flow cytometry, overcoming the key limitations of existing approaches for studying asymmetry during mitosis. ' 2011International Society for Advancement of Cytometry Key termsimaging flow cytometry; cell cycle analysis; asymmetric cell division; flow cytometry; image analysis DETERMINING how fate decisions are made at the molecular level is key to understanding how single cell progenitors can generate different specialized cell types. This diversification can occur in two ways. When a cell divides by mitosis, equal distribution of fate determining molecules across the plane of cytokinesis will produce two identical daughter cells that can differentiate in response to extrinsic stimuli. Alternatively, if fate determinants are apportioned asymmetrically prior to cytokinesis, two qualitatively and quantitatively distinct progeny will be generated by division (1,2). Asymmetric division is a key process for driving cellular diversity in developmental biology (3-7), stem cell biology (4,(8)(9)(10) and also recently in...

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Section: Methodssupporting

confidence: 72%

An imaging flow cytometric method for measuring cell division history and molecular symmetry during mitosis

et al. 2011

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“…To understand whether increased BrdU incorporation in ApoEdeficient dentate gyrus is due to the lengthening of S-phase or not, we measured the S-phase length of wild-type and ApoE-deficient neural stem and progenitor cells in vitro based on a validated protocol (Eidukevicius et al, 2005). We observed no differences in S-phase length between wild-type and ApoE-deficient neural progenitors (see Fig.…”

Section: Research Articlementioning

confidence: 95%

“…Cells were isolated into single cells and fixed in 70% ice-cold ethanol after the PBS wash. After BrdU staining, cells underwent flow cytometry analysis. S-phase length calculation is based on a previously validated method (Eidukevicius et al, 2005).…”

Section: S-phase Length Determinationmentioning

confidence: 99%

ApoE is required for maintenance of the dentate gyrus neural progenitor pool

et al. 2011

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SUMMARYMany genes regulating adult neurogenesis have been identified and are known to play similar roles during early neuronal development. We recently identified apolipoprotein E (ApoE) as a gene the expression of which is essentially absent in early brain progenitors but becomes markedly upregulated in adult dentate gyrus stem/progenitor cells. Here, we demonstrate that ApoE deficiency impairs adult dentate gyrus development by affecting the neural progenitor pool over time. We utilized ApoEdeficient mice crossed to a nestin-GFP reporter to demonstrate that dentate gyrus progenitor cells proliferate more rapidly at early ages, which is subsequently accompanied by an overall decrease in neural progenitor cell number at later time points. This appears to be secondary to over-proliferation early in life and ultimate depletion of the Type 1 nestin-and GFAP-expressing neural stem cells. We also rescue the proliferation phenotype with an ApoE-expressing retrovirus, demonstrating that ApoE works directly in this regard. These data provide novel insight into late hippocampal development and suggest a possible role for ApoE in neurodegenerative diseases.

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“…Proliferation was determined with the BD FITC BrdU Flow Kit by FACS, according to the recommendations by the manufacturer (48).…”

Section: Cd4mentioning

confidence: 99%

Ginger Phenylpropanoids Inhibit IL-1β and Prostanoid Secretion and Disrupt Arachidonate-Phospholipid Remodeling by Targeting Phospholipases A2

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Marazzi

Schoop³

et al. 2011

The Journal of Immunology

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The rhizome of ginger (Zingiber officinale) is employed in Asian traditional medicine to treat mild forms of rheumatoid arthritis and fever. We have profiled ginger constituents for robust effects on proinflammatory signaling and cytokine expression in a validated assay using human whole blood. Independent of the stimulus used (LPS, PMA, anti-CD28 Ab, anti-CD3 Ab, and thapsigargin), ginger constituents potently and specifically inhibited IL-1β expression in monocytes/macrophages. Both the calcium-independent phospholipase A2 (iPLA2)-triggered maturation and the cytosolic phospholipase A2 (cPLA2)-dependent secretion of IL-1β from isolated human monocytes were inhibited. In a fluorescence-coupled PLA2 assay, most major ginger phenylpropanoids directly inhibited i/cPLA2 from U937 macrophages, but not hog pancreas secretory phospholipase A2. The effects of the ginger constituents were additive and the potency comparable to the mechanism-based inhibitor bromoenol lactone for iPLA2 and methyl arachidonyl fluorophosphonate for cPLA2, with 10-gingerol/-shogaol being most effective. Furthermore, a ginger extract (2 μg/ml) and 10-shogaol (2 μM) potently inhibited the release of PGE2 and thromboxane B2 (>50%) and partially also leukotriene B4 in LPS-stimulated macrophages. Intriguingly, the total cellular arachidonic acid was increased 2- to 3-fold in U937 cells under all experimental conditions. Our data show that the concurrent inhibition of iPLA2 and prostanoid production causes an accumulation of free intracellular arachidonic acid by disrupting the phospholipid deacylation-reacylation cycle. The inhibition of i/cPLA2, the resulting attenuation of IL-1β secretion, and the simultaneous inhibition of prostanoid production by common ginger phenylpropanoids uncover a new anti-inflammatory molecular mechanism of dietary ginger that may be exploited therapeutically.

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A method to estimate cell cycle time and growth fraction using bromodeoxyuridine-flow cytometry data from a single sample

Cited by 23 publications

References 18 publications

An imaging flow cytometric method for measuring cell division history and molecular symmetry during mitosis

An imaging flow cytometric method for measuring cell division history and molecular symmetry during mitosis

ApoE is required for maintenance of the dentate gyrus neural progenitor pool

Ginger Phenylpropanoids Inhibit IL-1β and Prostanoid Secretion and Disrupt Arachidonate-Phospholipid Remodeling by Targeting Phospholipases A2

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