Assays based on Förster resonance energy transfer (FRET) can be used to study many processes in cell biology. Although this is most often done with microscopy for fluorescence detection, we report two ways to measure FRET in living cells by flow cytometry. Using a conventional flow cytometer and the "3-cube method" for intensity-based calculation of FRET efficiency, we measured the enzymatic activity of specific kinases in cells expressing a genetically-encoded reporter. For both AKT and protein kinase A, the method measured kinase activity in time-course, dose-response, and kinetic assays. Using the Cytek Aurora spectral flow cytometer, which applies linear unmixing to emission measured in multiple wavelength ranges, FRET from the same reporters was measured with greater single-cell precision, in real time and in the presence of other fluorophores. Results from gene-knockout studies suggested that spectral flow cytometry might enable the sorting of cells on the basis of FRET. The methods we present provide convenient and flexible options for using FRET with flow cytometry in studies of cell biology.

• Klíčová slova
• FRET, cell-based reporter assay, flow cytometry, kinase assay, protein kinase A, protein kinase B/AKT, spectral flow cytometry,
• MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   MeSH
• protoonkogenní proteiny c-akt * metabolismus   MeSH
• průtoková cytometrie metody   MeSH
• rezonanční přenos fluorescenční energie * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• luminescentní proteiny MeSH
• proteinkinasy závislé na cyklickém AMP MeSH
• protoonkogenní proteiny c-akt * MeSH

Cells across biological kingdoms release extracellular vesicles (EVs) as a means of communication with other cells, be their friends or foes. This is indeed true for the intracellular malaria parasite Plasmodium falciparum (Pf), which utilizes EVs to transport bioactive molecules to various human host systems. Yet, the study of this mode of communication in malaria research is currently constrained due to limitations in high-resolution tools and the absence of commercial antibodies. Here, we demonstrate the power of an advanced spectral flow cytometry approach to robustly detect secreted EVs, isolated from Pf-infected red blood cells. By labeling both EV membrane lipids and the DNA cargo within (non-antibody staining approach), we were able to detect a subpopulation of parasitic-derived EVs enriched in DNA. Furthermore, we could quantitatively measure the DNA-carrying EVs isolated from two distinct blood stages of the parasite: rings and trophozoites. Our findings showcase the potential of spectral flow cytometry to monitor dynamic changes in nucleic acid cargo within pathogenic EVs.

• Klíčová slova
• DNA, cargo, extracellular vesicles, flow cytometry, host pathogen, malaria, parasite,
• MeSH
• erytrocyty * parazitologie   metabolismus   MeSH
• extracelulární vezikuly * metabolismus   MeSH
• lidé MeSH
• Plasmodium falciparum * metabolismus   genetika   MeSH
• protozoální DNA * metabolismus   MeSH
• průtoková cytometrie * metody   MeSH
• tropická malárie * parazitologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protozoální DNA * MeSH

Herein, an advanced bioconjugation technique to synthesize hybrid polymer-antibody nanoprobes tailored for fluorescent cell barcoding in flow cytometry-based immunophenotyping of leukocytes is applied. A novel approach of attachment combining two fluorescent dyes on the copolymer precursor and its conjugation to antibody is employed to synthesize barcoded nanoprobes of antibody polymer dyes allowing up to six nanoprobes to be resolved in two-dimensional cytometry analysis. The major advantage of these nanoprobes is the construct design in which the selected antibody is labeled with an advanced copolymer bearing two types of fluorophores in different molar ratios. The cells after antibody recognition and binding to the target antigen have a characteristic double fluorescence signal for each nanoprobe providing a unique position on the dot plot, thus allowing antibody-based barcoding of cellular samples in flow cytometry assays. This technique is valuable for cellular assays that require low intersample variability and is demonstrated by the live cell barcoding of clinical samples with B cell abnormalities. In total, the samples from six various donors were successfully barcoded using only two detection channels. This barcoding of clinical samples enables sample preparation and measurement in a single tube.

• Klíčová slova
• antibody-polymer-dye conjugates, cellular barcoding, double fluorescence, flow cytometry, nanoprobes,
• MeSH
• fluorescenční barviva * chemie   MeSH
• imunofenotypizace MeSH
• polymery MeSH
• protilátky * MeSH
• průtoková cytometrie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fluorescenční barviva * MeSH
• polymery MeSH
• protilátky * MeSH

There is a wide range of techniques utilizing fluorescence of doxorubicin (Dox) commonly used for analysis of intracellular accumulation and destiny of various drug delivery systems containing this anthracycline antibiotic. Unfortunately, results of these studies can be significantly influenced by doxorubicin degradation product, 7,8-dehydro-9,10-desacetyldoxorubicinone (D*) forming spontaneously in aqueous environment, whose fluorescence strongly interfere with that of doxorubicin. Here, we define two microscopy techniques enabling to distinguish and separate Dox and D* emission based either on its spectral properties or on fluorescence lifetime analysis. To analyze influx and nuclear accumulation of Dox (free or polymer-bound) by flow cytometry, we propose using an indirect method based on its DNA intercalation competition with Hoechst 33342 rather than a direct measurement of doxorubicin fluorescence inside the cells.

• MeSH
• buněčné jádro metabolismus   MeSH
• buňky 3T3 MeSH
• DNA metabolismus   MeSH
• doxorubicin analogy a deriváty   metabolismus   farmakologie   MeSH
• fluorescenční spektrometrie MeSH
• hydrofobní a hydrofilní interakce MeSH
• lékové transportní systémy * MeSH
• lidé MeSH
• lymfom T-buněčný farmakoterapie   metabolismus   MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• polymery metabolismus   MeSH
• protinádorová antibiotika metabolismus   farmakologie   MeSH
• průtoková cytometrie MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 7,8-dehydro-9,10-desacetyldoxorubicinone MeSH Prohlížeč
• DNA MeSH
• doxorubicin MeSH
• polymery MeSH
• protinádorová antibiotika MeSH

BACKGROUND INFORMATION: Macarpine (MA) is a quaternary benzophenanthridine plant alkaloid isolated from Macleaya microcarpa or Stylophorum lasiocarpum. Benzophenanthridine alkaloids are interesting natural products that display antiproliferative, antimicrobial, antifungal and anti-inflammatory activities, and also fluorescence properties. In a previous study, we demonstrated that thanks to its ability to interact with DNA and its spectral properties MA could be used as a supravital DNA probe for fluorescence microscopy and flow cytometry including analyses of the cell cycle. In this study, we evaluated the suitability of MA as a DNA dye for time-lapse microscopy and flow-cytometric cell sorting. RESULTS: Living A-375 and MEF cells stained with MA were monitored by time-lapse microscopy for 24 h. Mitoses were observed at MA concentrations up to 0.5 μg/ml during the first 2-3 h. After this period of time, cells treated with MA at concentrations of 0.75 and 0.5 μg/ml underwent apoptosis. Cells cultivated with MA at concentration of 0.25 μg/ml or lower survived throughout the 24 h period. Toxicity of MA was dependent on light wavelength and frequency of image capturing. The intensity of MA fluorescence decreased during the incubation. MA concentration of 0.1 μg/ml was identified as the most suitable for live cell imaging with respect to fluorescence intensity and toxicity. MA at the concentration 10 μg/ml was used for sorting of enhanced green fluorescent protein (EGFP)-labelled neurons and fibroblasts yielding profiles similar to those obtained with DRAQ5. Contrary to DRAQ5, MA-stained cells survived in culture, and the sorted cells lost the MA signal suggesting reversible binding of the dye to the DNA. CONCLUSION: The results proved that MA may readily be used for chromosomes depicting and mitosis monitoring by time-lapse microscopy. In addition, MA has shown to be a suitable probe for sorting of EGFP-labelled cells, including neurons, that survived the labelling process. SIGNIFICANCE: In consideration of the results, we highly anticipate an onward use of MA in a broad range of applications based on live cell sorting and imaging, for example, cell synchronisation and monitoring of proliferation as an important experimental and/or diagnostic utility.

• Klíčová slova
• Cell cycle, DNA dye, FACS, Macarpine, Time-lapse imaging,
• MeSH
• benzofenantridiny analýza   MeSH
• buněčné kultury MeSH
• buněčný cyklus fyziologie   MeSH
• DNA analýza   MeSH
• fluorescenční barviva analýza   MeSH
• fluorescenční mikroskopie metody   MeSH
• lidé MeSH
• průtoková cytometrie * metody   MeSH
• separace buněk metody   MeSH
• viabilita buněk MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• benzofenantridiny MeSH
• DNA MeSH
• enhanced green fluorescent protein MeSH Prohlížeč
• fluorescenční barviva MeSH
• macarpine MeSH Prohlížeč
• zelené fluorescenční proteiny MeSH

A critical component of the EuroFlow standardization of leukemia/lymphoma immunophenotyping is instrument setup. Initially, the EuroFlow consortium developed a step-by-step standard operating protocol for instrument setup of ≥8-color flow cytometers that were available in 2006, when the EuroFlow activities started. Currently, there are 14 instruments from 9 manufacturers capable of 3-laser excitation and ≥8 color measurements. The specific adaptations required in the instrument set-up to enable them to acquire the standardized 8-color EuroFlow protocols are described here. Overall, all 14 instruments can be fitted with similar violet, blue and red lasers for simultaneous measurements of ≥8 fluorescent dyes. Since individual instruments differ both on their dynamic range (scale) and emission filters, it is not accurate to simply recalculate the target values to different scale, but adjustment of PMT voltages to a given emission filter and fluorochrome, is essential. For this purpose, EuroFlow has developed an approach using Type IIB (spectrally matching) particles to set-up standardized and fully comparable fluorescence measurements, in instruments from different manufacturers, as demonstrated here for the FACSCanto II, and Navios and MACSQuant flow cytometers. Data acquired after such adjustment on any of the tested cytometry platforms could be fully superimposed and therefore analyzed together. The proposed approach can be used to derive target values for any combination of spectrally distinct fluorochromes and any distinct emission filter of any new flow cytometry platform, which enables the measurement of the 8-color EuroFlow panels in a standardized way, by creating superimposable datafiles.

• Klíčová slova
• EuroFlow, Flow cytometry, Fluorescent spectrum, Fluorochromes, Standardization,
• MeSH
• hematologické nádory diagnóza   MeSH
• imunofenotypizace přístrojové vybavení   normy   MeSH
• lidé MeSH
• průtoková cytometrie přístrojové vybavení   normy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Quaternary benzo[c]phenanthridine alkaloids (QBAs) are naturally occurring compounds isolated from plants in the Fumariaceae, Papaveraceae, Ranunculaceae, and Rutaceae families. In addition to having a wide range of biological activities, they are also attractive for their fluorescent properties. We observed interesting fluorescent characteristics in the QBAs-macarpine (MA), sanguirubine (SR), chelirubine (CHR), sanguilutine (SL), chelilutine (CHL), sanguinarine (SA) and chelerythrine (CHE) after interaction with living cells. METHODS: Water stock solutions of the alkaloids (10-100 microg/ml) were added to intact cells, and after a brief incubation the cells were observed. Human cell lines HL60 (human promyelocytic leukemia), HeLa (human cervix adenocarcinoma), and LEP (human lung fibroblasts), and piglet blood were used in the experiments. Blood cells were stained with MA in combination with FITC-conjugated anti-CD45 surface marker antibody. Cells were analyzed by fluorescence microscopy and by flow cytometry. RESULTS: All tested alkaloids immediately entered living cells with MA, CHR, and SA binding to DNA. MA showed the best DNA staining properties. Fluorescence microscopy of MA, CHR, and SA stained cells described the nuclear architecture and clearly described chromosomes and apoptotic fragments in living cells. Moreover MA can rapidly represent the cellular DNA content of living cells at a resolution adequate for cell cycle analysis. QBAs were excitable using common argon lasers (488 nm) emitting at a range of 575-755 nm (i.e. fluorescence detectors FL2-5). Spectral characteristics of MA allow simultaneous surface immunophenotyping. CONCLUSIONS: It was shown that MA, CHR, and SA stain nucleic acids in living cells. They can be used as supravital fluorescent DNA probes, both in fluorescence microscopy and flow cytometry, including multiparameter analysis of peripheral blood and bone marrow. MA binds DNA stochiometrically and can provide information on DNA content.

• MeSH
• alkaloidy * chemie   metabolismus   MeSH
• DNA sondy * metabolismus   MeSH
• fluorescenční barviva MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• prasata MeSH
• průtoková cytometrie metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alkaloidy * MeSH
• DNA sondy * MeSH
• fluorescenční barviva MeSH

Schimke immuno-osseous dysplasia is a rare multisystemic disorder caused by biallelic loss of function of the SMARCAL1 gene that plays a pivotal role in replication fork stabilization and thus DNA repair. Individuals affected from this disease suffer from disproportionate growth failure, steroid resistant nephrotic syndrome leading to renal failure and primary immunodeficiency mediated by T cell lymphopenia. With infectious complications being the leading cause of death in this disease, researching the nature of the immunodeficiency is crucial, particularly as the state is exacerbated by loss of antibodies due to nephrotic syndrome or immunosuppressive treatment. Building on previous findings that identified the loss of IL-7 receptor expression as a possible cause of the immunodeficiency and increased sensitivity to radiation-induced damage, we have employed spectral cytometry and multiplex RNA-sequencing to assess the phenotype and function of T cells ex-vivo and to study changes induced by in-vitro UV irradiation and reaction of cells to the presence of IL-7. Our findings highlight the mature phenotype of T cells with proinflammatory Th1 skew and signs of exhaustion and lack of response to IL-7. UV light irradiation caused a severe increase in the apoptosis of T cells, however the expression of the genes related to immune response and regulation remained surprisingly similar to healthy cells. Due to the disease's rarity, more studies will be necessary for complete understanding of this unique immunodeficiency.

• Klíčová slova
• Cytometry, Exhaustion, IL-7, Immunodeficiency, Lymphopenia, Peritoneal dialysis, RNA-seq, SIOD, Schimke Imunoosseous dysplasia, Schimke immuno-osseous dysplasia, Spectral Cytometry, T cell, Thymus,
• MeSH
• apoptóza genetika   MeSH
• arterioskleróza genetika   etiologie   imunologie   MeSH
• dítě MeSH
• DNA-helikasy genetika   MeSH
• lidé MeSH
• metabolické nemoci kostí etiologie   genetika   MeSH
• nefrotický syndrom etiologie   genetika   MeSH
• oprava DNA * genetika   MeSH
• osteochondrodysplazie * genetika   imunologie   MeSH
• plicní embolie genetika   etiologie   MeSH
• poruchy růstu genetika   etiologie   MeSH
• primární imunodeficience * genetika   diagnóza   imunologie   MeSH
• syndromy imunologické nedostatečnosti genetika   imunologie   MeSH
• T-lymfocyty imunologie   MeSH
• ultrafialové záření škodlivé účinky   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA-helikasy MeSH
• SMARCAL1 protein, human MeSH Prohlížeč

The widely used fluorescent probe 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) serves as a pH-sensitive indicator in classical microscopy. Characteristics of BCECF were studied and a way of employing the probe in a confocal laser scanning microscope equipped with an argon laser at 488 nm was developed, based on the fact that the emission fluorescence spectra are pH-dependent with spectral maximum shift from 518 to 529 nm. Optical filters for the dual-emission ratio method were set to 506 and 529 nm. pH values measured inside a single cell of Saccharomyces cerevisiae were similar to those obtained with other pH estimation methods.

• MeSH
• argon MeSH
• fluoresceiny * MeSH
• fluorescenční barviva * MeSH
• fluorescenční spektrometrie MeSH
• intracelulární tekutina metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• konfokální mikroskopie metody   MeSH
• lasery MeSH
• průtoková cytometrie metody   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein MeSH Prohlížeč
• argon MeSH
• fluoresceiny * MeSH
• fluorescenční barviva * MeSH

The aim of this study was to investigate the impact of thymic dysplasia on the phenotypic and functional characteristics of T cells in patients with 22q11.2 deletion syndrome, including T-cell phenotype, transcriptional profile, cytokine production, as well as the possibility of utilizing IL-7 to recover their numbers and function. We found a strong bias towards Th1 response in pediatric and young adult 22q11.2DS patients, expansion of CXCR5+ follicular helper cells and CXCR3+CCR6- Th1 cells, increased production of cytokines IFN-γ, IL-10, IL-2, IL-21 and TNF-α. This Th1 skew was primarily driven by expanded terminally differentiated T cells. IL-7 further reduced naive T cells, increased cytokine production and caused an upregulation of exhaustion markers. Thus, Th1 bias in T cell populations persists from infancy into adolescence and is accompanied by accelerated maturation of T cells into memory stages. This phenotype is exacerbated by IL-7 which causes further decrease in naïve T cells in vitro.

• Klíčová slova
• Exhaustion, IFN-γ, IL-7, Immunodeficiency, RNA-seq, Spectral cytometry, thymus,
• MeSH
• cytokiny MeSH
• DiGeorgeův syndrom * MeSH
• dítě MeSH
• interferon gama * MeSH
• interleukin-7 MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• paměťové T-buňky MeSH
• Th1 buňky MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokiny MeSH
• interferon gama * MeSH
• interleukin-7 MeSH