Correct determination of the instantaneous level and changes of relevant proteins inside individual cells is essential for correct interpretation and understanding of physiological, diagnostic, and therapeutic events. Thus, single-cell analyses are important for quantification of natural cellular heterogeneity, which cannot be evaluated from averaged data of a cell population measurements. Here, we developed an original highly sensitive and selective instrumentation and methodology based on homogeneous single-step bioluminescence assay to quantify caspases and evaluate their heterogeneity in individual cells. Individual suspended cells are selected under microscope and reliably transferred into the 7 µl detection vials by a micromanipulator. The sensitivity of the method is given by implementation of photomultiplying tube with a cooled photocathode working in the photon counting mode. By optimization of our device and methodology, the limits of detection and quantitation were decreased down to 2.1 and 7.0 fg of recombinant caspase-3, respectively. These masses are lower than average amounts of caspase-3/7 in individual apoptotic and even non-apoptotic cells. As a proof of concept, the content of caspase-3/7 in single treated and untreated HeLa cells was determined to be 154 and 25 fg, respectively. Based on these results, we aim to use the technology for investigations of non-apoptotic functions of caspases.

• Klíčová slova
• Bioluminescence, Caspase detection and quantification, Photon counting, Single-cell analysis,
• MeSH
• apoptóza * MeSH
• HeLa buňky MeSH
• kaspasa 3 MeSH
• kaspasy * MeSH
• lidé MeSH
• technologie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kaspasa 3 MeSH
• kaspasy * MeSH

The protein heterogeneity at the single-cell level has been recognized to be vital for an understanding of various life processes during animal development. In addition, the knowledge of accurate quantity of relevant proteins at cellular level is essential for appropriate interpretation of diagnostic and therapeutic results. Some low-copy-number proteins are known to play a crucial role during cell proliferation, differentiation, and also in apoptosis. The fate decision is often based on the concentration of these proteins in the individual cells. This is likely to apply also for caspases, cysteine proteases traditionally associated with cell death via apoptosis but recently being discovered also as important factors in cell proliferation and differentiation. The hypothesis was tested in bone-related cells, where modulation of fate from apoptosis to proliferation/differentiation and vice versa is particularly challenging, e.g., towards anti-osteoporotic treatments and anti-cancer strategies. An ultrasensitive and highly selective method based on bioluminescence photon counting was used to quantify activated caspase-3/7 in order to demonstrate protein-level heterogeneity in individual cells within one population and to associate quantitative measurements with different cell fates (proliferation, differentiation, apoptosis). The results indicate a gradual increase of caspase-3/7 activation from the proliferative status to differentiation (more than three times) and towards apoptosis (more than six times). The findings clearly support one of the putative key mechanisms of non-apoptotic functions of pro-apoptotic caspases based on fine-tuning of their activation levels.

• Klíčová slova
• Apoptosis, Bioluminescence detection, Caspase-3/7, Cell death and differentiation, Single-cell detection and quantification,
• MeSH
• aktivace enzymů MeSH
• apoptóza MeSH
• buněčná diferenciace MeSH
• buněčné linie MeSH
• kaspasa 3 chemie   genetika   metabolismus   MeSH
• kaspasa 7 chemie   genetika   metabolismus   MeSH
• myši MeSH
• osteoblasty cytologie   fyziologie   MeSH
• proliferace buněk MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kaspasa 3 MeSH
• kaspasa 7 MeSH

Analysing the chemical content of individual cells has already been proven to reveal unique information on various biological processes. Single-cell analysis provides more accurate and reliable results for biology and medicine than analyses of extracts from cell populations, where a natural heterogeneity is averaged. To meet the requirements in the research of important biologically active molecules, such as caspases, we have developed a miniaturized device for simultaneous analyses of individual cells. A stainless steel body with a carousel holder enables high-sensitivity parallel detections in eight microvials. The holder is mounted in front of a photomultiplier tube with cooled photocathode working in photon counting mode. The detection of active caspase-3/7, central effector caspases in apoptosis, in single cells is based on the bioluminescence chemistry commercially available as Caspase-Glo® 3/7 reagent developed by Promega. Individual cells were captured from a culture medium under microscope and transferred by micromanipulator into detection microvial filled with the reagent. As a result of testing, the limits of detection and quantification were determined to be 0.27/0.86 of active caspase-3/7 content in an average apoptotic cell and 0.46/2.92 for non-apoptotic cells. Application potential of this technology in laboratory diagnostics and related medical research is discussed. Graphical abstract Miniaturized device for simultaneous analyses of individual cells.

• Klíčová slova
• Apoptosis, Bioluminescence, Caspase-3/7, Single-cell analysis,
• MeSH
• analýza jednotlivých buněk přístrojové vybavení   metody   MeSH
• apoptóza * MeSH
• design vybavení MeSH
• enzymatické testy přístrojové vybavení   metody   MeSH
• kaspasa 3 analýza   metabolismus   MeSH
• kaspasa 7 analýza   metabolismus   MeSH
• kultivované buňky MeSH
• luminiscenční měření přístrojové vybavení   metody   MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kaspasa 3 MeSH
• kaspasa 7 MeSH

Caspases are key enzymes activated during the apoptotic machinery. Apoptosis as a way of programmed cell death becomes deregulated in some pathologies including cancer transformations, neurodegenerative, or autoimmune diseases. Most of the methods available for the detection of apoptosis and caspases provide qualitative information only or quantification data as an average from cell populations or cell lysates. Several reports point to the importance of more accurate single-cell analyses in biomedical studies due to heterogeneity at tissue as well as cell level. To meet these requirements, we developed a miniaturized device enabling detection and quantification of active caspase-3/7 in individual cells at a femtogram level (10(-15) g). The active caspase-3/7 detection protocol is based on the bioluminescence chemistry commercially available as a Caspase-Glo™ 3/7 reagent developed by Promega. As a model, we used human stem cells treated by camptothecin to induce apoptosis. Individual apoptotic cells were captured from a culture medium under a microscope and transferred by a micromanipulation system into a detection capillary containing 2 μl of the reagent. Cells without activation by camptothecin served as negative controls. The detection limit of active caspase-3/7 achieved in the miniaturized system was determined as 0.20 and limit of quantification as 0.65 of the amount found in a single apoptotic human stem cell. Such a sensitive method could have a wide application potential in laboratory medicine and related clinically oriented research.

• MeSH
• analýza jednotlivých buněk přístrojové vybavení   MeSH
• apoptóza * MeSH
• buněčná diferenciace MeSH
• crista neuralis cytologie   MeSH
• design vybavení MeSH
• kamptothecin chemie   MeSH
• kaspasa 3 metabolismus   MeSH
• kaspasa 7 metabolismus   MeSH
• kmenové buňky účinky léků   patologie   MeSH
• lidé MeSH
• luminiscence MeSH
• mikromanipulace MeSH
• miniaturizace přístrojové vybavení   MeSH
• reprodukovatelnost výsledků MeSH
• zánět MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CASP3 protein, human MeSH Prohlížeč
• CASP7 protein, human MeSH Prohlížeč
• kamptothecin MeSH
• kaspasa 3 MeSH
• kaspasa 7 MeSH

A multi-FRET three-fluorophore probe containing coumarin, fluorescein and rhodamine B with two enzymatically cleavable linkers has been synthesized and optimized for the simultaneous activity detection and relative quantification of two proteases - caspase-8 and caspase-9. The probe designed as a ratiometric single-excitation triple-emission system shows specific change in fluorescence intensities upon enzymatic cleavage of individual linkers in model mixtures as well as in a cell lysate. The activation of caspase-8 and caspase-9 is responsible for initiation of extrinsic or intrinsic apoptotic pathway, respectively, and the probe was proposed as a single chemical tool which could help to decipher a mechanism of cell death induced by various stimuli. The main advantage of this probe is the simplicity of its preparation using conventional organic synthesis, easy application for measurement and evaluation of the results.

• Klíčová slova
• Caspases, FRET, Fluorescence, Fluorescent probe, Mathematical model, Proteases,
• MeSH
• aktivace enzymů MeSH
• apoptóza MeSH
• fluorescenční barviva * chemická syntéza   chemie   MeSH
• kaspasa 8 * analýza   metabolismus   MeSH
• kaspasa 9 * analýza   metabolismus   MeSH
• rezonanční přenos fluorescenční energie * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fluorescenční barviva * MeSH
• kaspasa 8 * MeSH
• kaspasa 9 * MeSH

This work describes the method of a selective hydride generation-cryotrapping (HG-CT) coupled to an extremely sensitive but simple in-house assembled and designed atomic fluorescence spectrometry (AFS) instrument for determination of toxicologically important As species. Here, an advanced flame-in-gas-shield atomizer (FIGS) was interfaced to HG-CT and its performance was compared to a standard miniature diffusion flame (MDF) atomizer. A significant improvement both in sensitivity and baseline noise was found that was reflected in improved (4 times) limits of detection (LODs). The yielded LODs with the FIGS atomizer were 0.44, 0.74, 0.15, 0.17 and 0.67 ng L(-1) for arsenite, total inorganic, mono-, dimethylated As and trimethylarsine oxide, respectively. Moreover, the sensitivities with FIGS and MDF were equal for all As species, allowing for the possibility of single species standardization with arsenate standard for accurate quantification of all other As species. The accuracy of HG-CT-AFS with FIGS was verified by speciation analysis in two samples of bottled drinking water and certified reference materials, NRC CASS-5 (nearshore seawater) and SLRS-5 (river water) that contain traces of methylated As species. As speciation was in agreement with results previously reported and sums of all quantified species corresponded with the certified total As. The feasibility of HG-CT-AFS with FIGS was also demonstrated by the speciation analysis in microsamples of exfoliated bladder epithelial cells isolated from human urine. The results for the sums of trivalent and pentavalent As species corresponded well with the reference results obtained by HG-CT-ICPMS (inductively coupled plasma mass spectrometry).

• MeSH
• arsen analýza   chemie   MeSH
• chemické techniky analytické ekonomika   přístrojové vybavení   MeSH
• fluorescenční spektrometrie normy   MeSH
• limita detekce MeSH
• nebulizátory a vaporizátory MeSH
• pitná voda chemie   MeSH
• spektrofotometrie atomová normy   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• arsen MeSH
• pitná voda MeSH

Macroautophagy is often quantified by live imaging of autophagosomes labeled with fluorescently tagged ATG8 protein (FP-ATG8) in Arabidopsis thaliana. The labeled particles are then counted in single focal planes. This approach may lead to inaccurate results as the actual 3D distribution of autophagosomes is not taken into account and appropriate sampling in the Z-direction is not performed. To overcome this issue, we developed a workflow consisting of immunolabeling of autophagosomes with an anti-ATG8 antibody followed by stereological image analysis using the optical disector and the Cavalieri principle. Our protocol specifically recognized autophagosomes in epidermal cells of Arabidopsis root. Since the anti-ATG8 antibody recognizes multiple AtATG8 isoforms, we were able to detect a higher number of immunolabeled autophagosomes than with the FP-AtATG8e marker, that most probably does not recognize all autophagosomes in a cell. The number of autophagosomes per tissue volume positively correlated with the intensity of autophagy induction. Compared with the quantification of autophagosomes in maximum intensity projections, stereological methods were able to detect the autophagosomes present in a given volume with higher accuracy. Our novel workflow provides a powerful toolkit for unbiased and reproducible quantification of autophagosomes and offers a convenient alternative to the standard of live imaging with FP-ATG8 markers.

• Klíčová slova
• ATG8, Cavalieri principle, autophagosome, autophagy, image analysis, immunofluorescence, immunolabeling, microscopy, optical disector, stereology,
• MeSH
• Arabidopsis * metabolismus   MeSH
• autofagie MeSH
• autofagozomy * metabolismus   MeSH
• kořeny rostlin * metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• průběh práce MeSH
• rodina proteinů Atg8 metabolismus   genetika   MeSH
• zobrazování trojrozměrné metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny huseníčku MeSH
• rodina proteinů Atg8 MeSH

Reverse transcription quantitative PCR (RT-qPCR) is already an established tool for mRNA detection and quantification. Since recently, this technique has been successfully employed for gene expression analyses, and also in individual cells (single cell RT-qPCR). Although the advantages of single cell measurements have been proven several times, a study correlating the expression measured on single cells, and in bulk samples consisting of a large number of cells, has been missing. Here, we collected a large data set to explore the relation between gene expression measured in single cells and in bulk samples, reflected by qPCR Cq values. We measured the expression of 95 genes in 12 bulk samples, each containing thousands of astrocytes, and also in 693 individual astrocytes. Combining the data, we described the relation between Cq values measured in bulk samples with either the percentage of the single cells that express the given genes, or the average expression of the genes across the single cells. We show that data obtained with single cell RT-qPCR are fully consistent with measurements in bulk samples. Our results further provide a base for quality control in single cell expression profiling, and bring new insights into the biological process of cellular expression.

• MeSH
• analýza jednotlivých buněk * MeSH
• gliový fibrilární kyselý protein genetika   MeSH
• messenger RNA genetika   MeSH
• myši transgenní MeSH
• myši MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• stanovení celkové genové exprese * MeSH
• zelené fluorescenční proteiny genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• enhanced green fluorescent protein MeSH Prohlížeč
• gliový fibrilární kyselý protein MeSH
• messenger RNA MeSH
• zelené fluorescenční proteiny MeSH

We describe here a new reversed-phase high-performance liquid chromatography with mass spectrometry detection method for quantifying intact cytokinin nucleotides in human K-562 leukemia cells. Tandem mass spectrometry was used to identify the intracellular metabolites (cytokinin monophosphorylated, diphosphorylated, and triphosphorylated nucleotides) in riboside-treated cells. For the protein precipitation and sample preparation, a trichloroacetic acid extraction method is used. Samples are then back-extracted with diethyl ether, lyophilized, reconstituted, and injected into the LC system. Analytes were quantified in negative selected ion monitoring mode using a single quadrupole mass spectrometer. The method was validated in terms of retention time stabilities, limits of detection, linearity, recovery, and analytical accuracy. The developed method was linear in the range of 1-1,000 pmol for all studied compounds. The limits of detection for the analytes vary from 0.2 to 0.6 pmol.

• MeSH
• chromatografie kapalinová * MeSH
• cytokininy analýza   chemie   MeSH
• hmotnostní spektrometrie * MeSH
• lidé MeSH
• molekulární struktura MeSH
• nádorové buněčné linie MeSH
• nukleotidy analýza   chemie   MeSH
• tandemová hmotnostní spektrometrie * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokininy MeSH
• nukleotidy MeSH

The human p73 protein is essential for normal morphogenesis and maintenance of neural tissue. Recently, several TP73 transcripts have been revealed in medulloblastoma (MB), the most common malignant brain tumor in children. Here, we performed immunohistochemical analysis on 29 MB specimens using anti-p73alpha and anti-DeltaNp73 antibodies. Real-time PCR quantification was performed to assess TAp73 and DeltaNp73 transcripts in a subset of 13 MB samples. Normal cerebellar tissues and RNA were used for comparison. Pilot clinical-pathological correlations were also provided. We report significant differences for TAp73 and DeltaNp73 mRNA expression between tumor tissues and reference (P = 0.013, P = 0.028). Immunohistochemically, 52 and 29% MB samples were positive for p73alpha and DeltaNp73, respectively. p73alpha expression was found to be in both the nucleus and cytoplasm, whereas DeltaNp73 was localized predominantly in the cytoplasm. In normal cerebellum, positive staining for p73alpha and DeltaNp73 was observed in the Purkinje cells of newborns, not adult samples, which supports the developmental role of TP73 during organogenesis of the human cerebellum. Survival analysis has shown negative relationship of DeltaNp73-immunoreactivity with overall survival (OS) and event free survival (EFS) (P = 0.026 and P = 0.127, respectively). For p73alpha-positive cases, the negative trend in OS (P = 0.149) and EFS (P = 0.216) was also apparent. Our results indicate the involvement of p73 protein in MB tumorigenesis and define TP73 as a potential prognostic and therapeutic target for medulloblastoma.

• MeSH
• dítě MeSH
• DNA vazebné proteiny genetika   imunologie   metabolismus   MeSH
• dospělí MeSH
• imunohistochemie MeSH
• jaderné proteiny genetika   imunologie   metabolismus   MeSH
• lidé MeSH
• meduloblastom genetika   metabolismus   patofyziologie   MeSH
• messenger RNA analýza   metabolismus   MeSH
• míra přežití MeSH
• mladiství MeSH
• mozek metabolismus   patologie   patofyziologie   MeSH
• nádorová transformace buněk genetika   metabolismus   MeSH
• nádorové biomarkery analýza   genetika   metabolismus   MeSH
• nádorové supresorové proteiny genetika   imunologie   metabolismus   MeSH
• nádory mozku genetika   metabolismus   patofyziologie   MeSH
• předškolní dítě MeSH
• prognóza MeSH
• protein - isoformy genetika   izolace a purifikace   metabolismus   MeSH
• regulace genové exprese u nádorů genetika   MeSH
• retrospektivní studie MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• jaderné proteiny MeSH
• messenger RNA MeSH
• nádorové biomarkery MeSH
• nádorové supresorové proteiny MeSH
• protein - isoformy MeSH