BACKGROUND: Because of its non-destructive nature, label-free imaging is an important strategy for studying biological processes. However, routine microscopic techniques like phase contrast or DIC suffer from shadow-cast artifacts making automatic segmentation challenging. The aim of this study was to compare the segmentation efficacy of published steps of segmentation work-flow (image reconstruction, foreground segmentation, cell detection (seed-point extraction) and cell (instance) segmentation) on a dataset of the same cells from multiple contrast microscopic modalities. RESULTS: We built a collection of routines aimed at image segmentation of viable adherent cells grown on the culture dish acquired by phase contrast, differential interference contrast, Hoffman modulation contrast and quantitative phase imaging, and we performed a comprehensive comparison of available segmentation methods applicable for label-free data. We demonstrated that it is crucial to perform the image reconstruction step, enabling the use of segmentation methods originally not applicable on label-free images. Further we compared foreground segmentation methods (thresholding, feature-extraction, level-set, graph-cut, learning-based), seed-point extraction methods (Laplacian of Gaussians, radial symmetry and distance transform, iterative radial voting, maximally stable extremal region and learning-based) and single cell segmentation methods. We validated suitable set of methods for each microscopy modality and published them online. CONCLUSIONS: We demonstrate that image reconstruction step allows the use of segmentation methods not originally intended for label-free imaging. In addition to the comprehensive comparison of methods, raw and reconstructed annotated data and Matlab codes are provided.
- MeSH
- algoritmy MeSH
- frakcionace buněk metody MeSH
- lidé MeSH
- mikroskopie metody MeSH
- počítačové zpracování obrazu MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The aims of this study were to determine concentrations of total homocysteine, cysteine, cysteinylglycine and glutathione in spermatozoa, seminal fluid and blood plasma and to analyse their relationships with sperm parameters. For this reason, a new highly effective method of spermatozoa lysis was developed, using methanol, freezing and subsequent thawing in ultrasonic bath. An HPLC-FD assay was conducted on thiols concentrations in lysed spermatozoa, seminal fluid and blood plasma. Concentrations of thiols in spermatozoa were significantly lower in men with normozoospermia than in samples with pathological semen parameters. Statistical analysis found significant correlations between thiol concentrations in spermatozoa and semen parameters, while the same analysis with thiol concentrations in seminal fluid was substantially less powerful. Only cysteinylglycine concentrations in seminal fluid significantly correlated with pathological semen parameters. No significant differences or correlations were found with blood plasma concentrations.
- MeSH
- dospělí MeSH
- frakcionace buněk metody MeSH
- homocystein analýza krev MeSH
- intracelulární prostor chemie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mužská infertilita krev metabolismus MeSH
- sperma chemie MeSH
- spermie chemie ultrastruktura MeSH
- sulfhydrylové sloučeniny analýza krev MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
A synchronous population of cells is one of the prerequisites for studying cell cycle processes such as DNA replication, nuclear and cellular division. Green algae dividing by multiple fission represent a unique single cell system enabling the preparation of highly synchronous cultures by application of a light-dark regime similar to what they experience in nature. This chapter provides detailed protocols for synchronization of different algal species by alternating light-dark cycles; all critical points are discussed extensively. Moreover, detailed information on basic analysis of cell cycle progression in such cultures is presented, including analyses of nuclear, cellular, and chloroplast divisions. Modifications of basic protocols that enable changes in cell cycle progression are also suggested so that nuclear or chloroplast divisions can be followed separately.
- MeSH
- barvení a značení metody MeSH
- buněčné dělení MeSH
- buněčné kultury metody MeSH
- buněčný cyklus MeSH
- Chlamydomonas reinhardtii cytologie genetika růst a vývoj MeSH
- Chlorophyta cytologie genetika růst a vývoj MeSH
- chloroplasty genetika MeSH
- DNA rostlinná genetika MeSH
- fotoperioda * MeSH
- frakcionace buněk metody MeSH
- replikace DNA MeSH
- světlo MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The initiation of T-cell receptor (TCR) signaling, based on the cobinding of TCR and CD4-Lck heterodimer to a peptide-major histocompatibility complex II on antigen presenting cells, represents a classical model of T-cell signaling. What is less clear however, is the mechanism which translates TCR engagement to the phosphorylation of immunoreceptor tyrosine-based activation motifs on CD3 chains and how this event is coupled to the delivery of Lck function. Recently proposed 'standby model of Lck' posits that resting T-cells contain an abundant pool of constitutively active Lck (pY394(Lck)) required for TCR triggering, and this amount, upon TCR engagement, remains constant. Here, we show that although maintenance of the limited pool of pY394(Lck) is necessary for the generation of TCR proximal signals in a time-restricted fashion, the total amount of this pool, ~2%, is much smaller than previously reported (~40%). We provide evidence that this dramatic discrepancy in the content of pY394(Lck)is likely the consequence of spontaneous phosphorylation of Lck that occurred after cell solubilization. Additional discrepancies can be accounted for by the sensitivity of different pY394(Lck)-specific antibodies and the type of detergents used. These data suggest that reagents and conditions used for the quantification of signaling parameters must be carefully validated and interpreted. Thus, the limited size of pY394(Lck) pool in primary T-cells invites a discussion regarding the adjustment of the quantitative parameters of the standby model of Lck and reevaluation of the mechanism by which this pool contributes to the generation of proximal TCR signaling.
- MeSH
- aktivace lymfocytů MeSH
- antigeny CD45 genetika metabolismus MeSH
- artefakty * MeSH
- benzochinony farmakologie MeSH
- fosforylace účinky léků MeSH
- frakcionace buněk metody MeSH
- Jurkat buňky MeSH
- lidé MeSH
- makrocyklické laktamy farmakologie MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- myši MeSH
- protein-tyrosinkináza ZAP-70 metabolismus MeSH
- receptory antigenů T-buněk metabolismus MeSH
- signální transdukce účinky léků MeSH
- T-lymfocyty imunologie MeSH
- tyrosinkinasa p56(lck), specifická pro lymfocyty metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Outer membrane vesicles secreted by gram-negative bacteria play an important role in bacterial physiology as well as in virulence and host-pathogen interaction. Isolated vesicles of some bacteria have also been studied for their immunomodulatory potential in the vaccine development. However, the production of vesicles in sufficient amount, purity and reproducibility remains a critical challenge for subsequent analyses in most bacteria. In the present review methods of production, isolation, purification and quantification of outer membrane vesicles are summarized and discussed.
- MeSH
- buněčná stěna * imunologie metabolismus MeSH
- frakcionace buněk metody MeSH
- fyziologický stres MeSH
- genetické inženýrství MeSH
- gramnegativní bakterie * genetika imunologie metabolismus MeSH
- sekreční vezikuly * imunologie metabolismus MeSH
- subcelulární frakce MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
We present a simple method for enrichment of lysosomal membranes from HEK293 and HeLa cell lines taking advantage of selective disruption of lysosomes by methionine methyl ester. Organelle concentrate from postnuclear supernatant was treated with 20 mmol/l methionine methyl ester for 45 min to lyse the lysosomes. Subsequently, lysosomal membranes were resolved on a step sucrose gradient. An enriched lysosomal membrane fraction was collected from the 20%/35% sucrose interface. The washed lysosomal membrane fraction was enriched 30 times relative to the homogenate and gave the yield of more than 8%. These results are comparable to lysosomal membranes isolated by magnetic chromatography from cultured cells (Diettrich et al., 1998). The procedure effectively eliminated mitochondrial contamination and minimized contamination from other cell compartments. The enriched fractions retained the ability to acidify membrane vesicles through the activity of lysosomal vacuolar ATPase. The method avoids non-physiological overloading of cells with superparamagnetic particles and appears to be quite robust among the tested cell lines. We expect it may be of more general use, adaptable to other cell lines and tissues.
- MeSH
- adenosintrifosfát farmakologie MeSH
- centrifugace - gradient hustoty MeSH
- frakcionace buněk metody MeSH
- glukosylceramidasa metabolismus MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- intracelulární membrány účinky léků metabolismus MeSH
- kyseliny metabolismus MeSH
- lidé MeSH
- lyzozomy účinky léků metabolismus MeSH
- subcelulární frakce účinky léků metabolismus MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The following bead mills used for disruption of the microalga Chlorella cells were tested: (1) Dyno-Mill ECM-Pilot, grinding chamber volume 1.5 L; KDL-Pilot A, chamber volume 1.4 L; KD 20 S, chamber volume 18.3 L; KD 25 S, chamber volume 26 L of Willy A. Bachofen, Basel, Switzerland, (2) LabStar LS 1, chamber volume 0.6 L of Netzsch, Selb, Germany, (3) MS 18, chamber volume 1.1 L of FrymaKoruma, Neuenburg, Germany. Amount of disrupted cells decreased with increasing Chlorella suspension feed rate and increased up to about 85% of the beads volume in the grinding chamber of the homogenizers. It also increased with agitator speed and number of passes of the algae suspension through the chamber. The optimum beads diameter was 0.3-0.5 mm in the homogenizers Dyno-Mill and LabStar LS 1 and 0.5-0.7 mm in the homogenizer MS 18. While the degree of the cell disruption decreased with increasing cell density in Dyno-Mill and LabStar, the cell disruption in the MS 18 increased. Depending on processing parameters, more than 90% of algae cells were disrupted by passing through the bead mills and bacteria count in algae suspension was reduced to about two orders.
- MeSH
- bakteriální infekce mikrobiologie patofyziologie MeSH
- buněčné linie mikrobiologie MeSH
- fagozomy * fyziologie mikrobiologie MeSH
- frakcionace buněk metody MeSH
- Francisella tularensis * patogenita MeSH
- interakce hostitele a patogenu * fyziologie MeSH
- kathepsin D izolace a purifikace MeSH
- makrofágy fyziologie mikrobiologie MeSH
- membránový protein 1 asociovaný s lyzozomy izolace a purifikace MeSH
- rab proteiny vázající GTP izolace a purifikace MeSH
- techniky in vitro MeSH
- Publikační typ
- práce podpořená grantem MeSH
