Fluorescence Labeling Of Cells Research Articles

Fluorescent carbon dots for ratiometric detection of curcumin and ferric ion based on inner filter effect, cell imaging and PVDF membrane fouling research of iron flocculants in wastewater treatment

The CDs were used as the ratiometric probe for detection of curcumin and Fe3+. The fluorescence of CDs at 420 nm was quenched through inner filter effect (IFE) while the fluorescence of curcumin at 525 nm was enhanced after adding curcumin. The value of ln(I525/I420) increased linearly with the curcumin concentration in the range of 5–30 μM, the limit of detection was 21.79 nM. After adding Fe3+ into the system of CDs-curcumin, curcumin complexed with Fe3+ leading to the quenching of the fluorescence of curcumin at 525 nm, which declined the spectra overlap between curcumin and CDs. The process of IFE was inhibited, the fluorescence of CDs at 420 nm was recovered. The value of ln(I525/I420) linearly decreased with the increasing of Fe3+ in the range of 2-14 μM, the limit of detection is 18.11 nM. The CDs-curcumin system was successfully applied for H1299 cell imaging and has potential applications in the fields of fluorescent labeling and inhibition of tumor cells. The CDs-curcumin system was furtherly used for PVDF membrane fouling research of iron flocculants in wastewater treatment.

Electrical measurement and quantification of different cell types by electro chemical impedance spectroscopy

Recently, in the field of regenerative tissue engineering, huge amount of cells are needed for developping new organs and tissues. However, during cell cultivation, cells sometimes show degeneration or change their original characters so that it is needed to separate those cells from original cells. Although fluorescent labeling and magnetic labeling of cells have been used for cell separation, invasive label-free methods are required from the point of view of cell damage. Therefore, our aim is to develop a high-efficiency label-free cell separation system based on the difference in electrical characteristics of cells. In this study, for its first step, an attempt was made to distinguish different type of cells by measuring their electrical properties.

Modified Intravital Microscopy to Assess Vascular Health and T-Cell Motility.

The ability to study the microcirculation in real time is key to elucidating how the immune system and the associated microvasculature interact and influence one another within the lymph node (LN). Here, we present a method for near in-situ imaging of the inguinal LN. In particular, this method is ideal for the assessment of overall vascular health that influences immune functions and for the evaluation of T-cell motility. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, quantification of vessel magnitude and vessel integrity. Modified intravital microscopy (M-IVM) of the LNs allows direct evaluation of microvascular functions as well as real-time imaging of the direct interface between immune cells, the LN, and the microcirculation. Importantly, M-IVM technique can be readily combined with many other vascular and immunological techniques such as fluorescent cell labeling and assessment of sticking and rolling time as descripted. Furthermore, it can be adapted to study vasculature of other than the inguinal LN. Overall, this chapter provides a dependable method for fundamental vascular immunological assessment of LNs that is decidedly useful in a diverse range of investigations.

Photo-responsive cyclodextrin/anthracene/Eu3+ supramolecular assembly for a tunable photochromic multicolor cell label and fluorescent ink.

A photo-responsive supramolecular assembly was successfully constructed through the stoichiometric 2 : 1 non-covalent association of two 4-(anthracen-2-yl)pyridine-2,6-dicarboxylic acid (1) units in one γ-cyclodextrin (γ-CD) cavity, followed by the subsequent coordination polymerization of the γ-CD·1 2 (1 2 = two 1) inclusion complex with Eu(iii). Interestingly, owing to the photodimerization behavior of anthracene units and the excellent luminescence properties of Eu(iii), the Eu3+⊂γ-CD·1 2 system showed multicolor fluorescence emission from cyan to red by irradiation for 0-16 minutes. Moreover, white light emission with CIE coordinates (0.32 and 0.36) was achieved at 4 min. Importantly, white light-containing multicolor emission could be obtained in water, solid films and living cells. Especially, the Eu3+⊂γ-CD·1 2 system could tag living cells with marvelous white fluorescence and display no obvious cytotoxicity. Thus, this supramolecular assembly offers a new pathway in the fields of tunable photochromic fluorescent ink and cell labelling.

Preparation of fluorescent waterborne polyurethane nanodispersion by high-gravity miniemulsion polymerization for multifunctional applications

Polymer encapsulated fluorescent nanoparticles have found many applications in bioimaging, fluorescent gels and inks. However, their translation into the practical products has been slow because it is still difficult to produce nanoparticles that are consistent “batch-to-batch” for scale-up. Herein, we develop a high-gravity miniemulsion polymerization route for fluorescent waterborne polyurethane (PU) by using rotating packed bed (RPB) reactor for process intensification. In optimized conditions, the PU nanoparticles obtained exhibited an average size of 137 nm with narrow size distribution. Dispersion stability of the PU nanodispersions was investigated and no significant sediments or aggregates were observed for up to one month. The 1,1,2,3,4,5-hexaphenylsilole dyes, a kind of typical aggregation-induced emission luminogens (AIEgens), could be in situ loaded in PU nanoparticles, forming fluorescent nanodispersions of AIEgens@PU in aqueous solution. The preliminary applications of AIEgens@PU nanodispersion for in vitro fluorescent labeling of cells, transparent fluorescent films and anti-counterfeit printing inks were demonstrated. This study demonstrated that the newly developed high-gravity miniemulsion polymerization method provides an effective route to waterborne PU nanodispersion for multifunctional applications.

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum.

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

Performance of Fluorescent Cell-Labeling Dyes under Simulated Europa Mission Radiation Doses.

We investigated the performance of several commonly used fluorescent dyes after exposure to a simulated Europa mission total ionizing radiation dose of 300 krad (3 kGy) applied using a 60Co source. Dyes irradiated in aqueous solution or as lyophilized powders were evaluated for absorbance and emission spectra, quantum yield, and where appropriate, ability to label cells or nucleic acids. Although some dyes showed significant increase or decrease in quantum yield with the dose, their spectra and cell-labeling properties remained essentially unchanged after irradiation in powder form. Irradiation in aqueous solution led to significantly greater changes, including a large blue shift in the DNA intercalator propidium iodide. These results suggest that many fluorescent probes are appropriate for use in astrobiological missions to Europa, but that SYTO9 and propidium iodide should be used with caution or not mixed with each other, as is commonly done in "Live/Dead" labeling applications.

A chemotactic model of trunk neural crest cell migration.

Trunk neural crest cells follow a common ventral migratory pathway but are distributed into two distinct locations to form discrete sympathetic and dorsal root ganglia along the vertebrate axis. Although fluorescent cell labeling and time-lapse studies have recorded complex trunk neural crest cell migratory behaviors, the signals that underlie this dynamic patterning remain unclear. The absence of molecular information has led to a number of mechanistic hypotheses for trunk neural crest cell migration. Here, we review recent data in support of three distinct mechanisms of trunk neural crest cell migration and develop and simulate a computational model based on chemotactic signaling. We show that by integrating the timing and spatial location of multiple chemotactic signals, trunk neural crest cells may be accurately positioned into two distinct targets that correspond to the sympathetic and dorsal root ganglia. In doing so, we honor the contributions of Wilhelm His to his identification of the neural crest and extend the observations of His and others to better understand a complex question in neural crest cell biology.

Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase

Glucose oxidase (GOx) mutants with higher activity or stability have important role in industry and in the development of biosensors and biofuel cells. Discovering these mutants can be time-consuming if appropriate high-throughput screening (HTS) systems are not available. GOx gene libraries were successfully screened and sorted using a HTS system based on GOx activity dependent fluorescent labeling of yeast cells with tyramids and quantification of the amount of expressed enzyme by yeast enhanced green fluorescent protein (yGFP) tagging and flow cytometry. For this purpose, we expressed wild type and a mutant GOx as a chimera with the yGFP to confirm differences in catalytic activity between wild-type and mutant GOx. Fluorescence of yGFP is preserved during expression of chimera, and also after the oxidative enzymatic reaction. We have obtained a 2.5-fold enrichment in population of cells expressing active enzyme, and percentage of enzyme variants with enzymatic mean activity higher than wild type activity was increased to 44% after a single round of GOx gene library sorting. We have found two mutants with 1.3 and 2.3-fold increase in Vmax values compared to the wtGOx. By simultaneous detection of protein expression level and enzyme activity we have increased the likelihood of finding GOx variants with increased activity in a single round of flow cytometry sorting.

Polycondensation of a Perylene Bisimide Derivative and L-Malic Acid as Water-Soluble Conjugates for Fluorescent Labeling of Live Mammalian Cells

Based on simple mixing and polymerization of a hydroxyl-containing derivative of perylene bisimide (PBI) and l-malic acid, here, we demonstrate a new type of dye-polymer conjugate, PBI-poly(α,β-malic acid) (PBI–PMA). Benefiting from the excellent water-solubility of weak polyanionic PMA structure and the high fluorescence of PBI, the PBI-PMA conjugates readily dissolve in water, displaying strong pH-dependent fluorescence with the highest intensity at pH 6. Due to the excellent biocompatibility of PMA, those conjugates showed low cytotoxicity on L929 cells. Using L929 and HeLa cells, we also confirmed that the PBI-PMA-labeled cells display intense fluorescence. Overall, the PBI-PMA conjugate demonstrates high potential as a cell labeling agent with its synthesis ease, good solubility in aqueous medium, low cytotoxicity, and high fluorescence.

Bicistronic 2A-peptide-based co-expression reporter knock-in strategy by CRISPR/Cas9 system: application to the labeling of specific cell lineages and gene expression monitoring

Fluorescent cell labeling is used to identify the specific cell lineages in a tissue or a whole organism. Transgenic organisms with fluorescent reporter genes have been created to visualize specific cell lineages and to investigate cell specific morphologies, motilities, gene expressions, neural activities, intracellular signaling, etc. However, in human cells, transgenes are often silenced during cell differentiation, and so knock-in technology was adopted to label the specific human cell lineages, although the establishment of knock-in human pluripotent stem cells (hPSCs) required considerable efforts. Genome editing technology paved the way to more efficient and useful knock-in methods. Also, we applied a bicistronic 2A-peptide-based co-expression (B2AC) system to the knock-in strategy for the fluorescent cell labeling. By using these technologies, knock-in hPSC lines were established, and the expression of Crx, a specific photoreceptor marker, was revealed during three-dimensional retinal organoid culture. The Crx expression and fluorescent intensity in the cells were positively correlated, suggesting that the B2AC reporter system functioned during human retinal development. The immunohistochemistry of Crx and the maturation of fluorescent reporter cells after long-term differentiation culture indicated that knock-in of the reporter gene did not affect the function of the target Crx gene. B2AC reporter cells successfully represented Crx upregulation by DAPT, a Notch signal inhibitor, during retinal differentiation from hPSC. These results indicated that the B2AC reporter knock-in system could be used to investigate cell transplantation, developmental mechanisms, disease signaling, drug screening, and intracellular signaling.

Subgroup Characteristics of Marine Methane-Oxidizing ANME-2 Archaea and Their Syntrophic Partners as Revealed by Integrated Multimodal Analytical Microscopy.

Phylogenetically diverse environmental ANME archaea and sulfate-reducing bacteria cooperatively catalyze the anaerobic oxidation of methane oxidation (AOM) in multicelled consortia within methane seep environments. To better understand these cells and their symbiotic associations, we applied a suite of electron microscopy approaches, including correlative fluorescence in situ hybridization-electron microscopy (FISH-EM), transmission electron microscopy (TEM), and serial block face scanning electron microscopy (SBEM) three-dimensional (3D) reconstructions. FISH-EM of methane seep-derived consortia revealed phylogenetic variability in terms of cell morphology, ultrastructure, and storage granules. Representatives of the ANME-2b clade, but not other ANME-2 groups, contained polyphosphate-like granules, while some bacteria associated with ANME-2a/2c contained two distinct phases of iron mineral chains resembling magnetosomes. 3D segmentation of two ANME-2 consortium types revealed cellular volumes of ANME and their symbiotic partners that were larger than previous estimates based on light microscopy. Polyphosphate-like granule-containing ANME (tentatively termed ANME-2b) were larger than both ANME with no granules and partner bacteria. This cell type was observed with up to 4 granules per cell, and the volume of the cell was larger in proportion to the number of granules inside it, but the percentage of the cell occupied by these granules did not vary with granule number. These results illuminate distinctions between ANME-2 archaeal lineages and partnering bacterial populations that are apparently unified in their ability to perform anaerobic methane oxidation.IMPORTANCE Methane oxidation in anaerobic environments can be accomplished by a number of archaeal groups, some of which live in syntrophic relationships with bacteria in structured consortia. Little is known of the distinguishing characteristics of these groups. Here, we applied imaging approaches to better understand the properties of these cells. We found unexpected morphological, structural, and volume variability of these uncultured groups by correlating fluorescence labeling of cells with electron microscopy observables.

Interspecific germ cell transplantation: a new light in the conservation of valuable Balkan trout genetic resources?

Interspecific transplantation of germ cells from the brown trout Salmo trutta m. fario and the European grayling Thymallus thymallus into rainbow trout Oncorhynchus mykiss recipients was carried out in order to improve current practices in conservation of genetic resources of endangered salmonid species in the Balkan Peninsula. Current conservation methods mainly include in situ efforts such as the maintenance of purebred individuals in isolated streams and restocking with purebred fingerlings; however, additional ex situ strategies such as surrogate production are needed. Steps required for transplantation such as isolation of high number of viable germ cells and fluorescent labeling of germ cells which are to be transplanted have been optimized. Isolated and labeled brown trout and grayling germ cells were intraperitoneally transplanted into 3 to 5days post hatch rainbow trout larvae. Survival of the injected larvae was comparable to the controls. Sixty days after transplantation, fluorescently labeled donor cells were detected within the recipient gonads indicating successful incorporation of germ cells (brown trout spermatogonia and oogonia-27%; grayling spermatogonia-28%; grayling oogonia-23%). PCR amplification of donor mtDNA CR fragments within the recipient gonads additionally corroborated the success of incorporation. Overall, the transplantation method demonstrated in this study presents the first step and a possible onset of the application of the germ cell transplantation technology in conservation and revitalization of genetic resources of endangered and endemic species or populations of salmonid fish and thus give rise to new or improved management strategies for such species.

Dystrophin Expressing Chimeric (DEC) Human Cells Provide a Potential Therapy for Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a progressive and lethal disease caused by mutations of the dystrophin gene. Currently no cure exists. Stem cell therapies targeting DMD are challenged by limited engraftment and rejection despite the use of immunosuppression. There is an urgent need to introduce new stem cell-based therapies that exhibit low allogenic profiles and improved cell engraftment. In this proof-of-concept study, we develop and test a new human stem cell-based approach to increase engraftment, limit rejection, and restore dystrophin expression in the mdx/scid mouse model of DMD. We introduce two Dystrophin Expressing Chimeric (DEC) cell lines created by ex vivo fusion of human myoblasts (MB) derived from two normal donors (MBN1/MBN2), and normal and DMD donors (MBN/MBDMD). The efficacy of fusion was confirmed by flow cytometry and confocal microscopy based on donor cell fluorescent labeling (PKH26/PKH67). In vitro, DEC displayed phenotype and genotype of donor parent cells, expressed dystrophin, and maintained proliferation and myogenic differentiation. In vivo, local delivery of both DEC lines (0.5 × 106) restored dystrophin expression (17.27%±8.05—MBN1/MBN2 and 23.79%±3.82—MBN/MBDMD) which correlated with significant improvement of muscle force, contraction and tolerance to fatigue at 90 days after DEC transplant to the gastrocnemius muscles (GM) of dystrophin-deficient mdx/scid mice. This study establishes DEC as a potential therapy for DMD and other types of muscular dystrophies.

Tissue-Clearing Techniques Enable Three-Dimensional Visualization of Aerosolized Model Compound and Lung Structure at the Alveolar Scale.

In this study, we examined the usefulness of a tissue-clearing technique for the evaluation of the lung distribution of aerosolized drugs. An aerosol formulation of TexasRed dextran (70 kDa), a model compound of drug carrier for aerosolized drugs, was administered intrapulmonarily to mice using a MicroSprayer, and then DyLight 488-conjugated tomato lectin was administered intravenously to visualize general lung structure via the fluorescent labeling of alveolar and bronchial epithelial cells. Tissue clearing followed by laser scanning confocal microscopy enabled the three-dimensional visualization of intrapulmonary TexasRed dextran and the evaluation of its distribution at the alveolar scale without the preparation of thin tissue sections. These findings suggest that tissue-clearing techniques are useful for the evaluation of intrapulmonary distribution and development of pulmonary drug delivery systems.

Quantum dot–Cramoll lectin as novel conjugates to glycobiology

The optical properties of quantum dots (QDs) make them useful tools for biology, especially when combined with biomolecules such as lectins. QDs conjugated to lectins can be used as nanoprobes for carbohydrate expression analysis, which can provide valuable information about glycosylation changes related to cancer and pathogenicity of microorganisms, for example. In this study, we evaluated the best strategy to conjugate Cramoll lectin to QDs and used the fluorescent labeling of Candida albicans cells as a proof-of-concept. Cramoll is a mannose/glucose–binding lectin with unique biological properties such as immunomodulatory, antiparasitic, and antitumor activities. We probed covalent coupling and adsorption as conjugation strategies at different pH values. QDs conjugated to Cramoll at pH7.0 showed the best labeling efficiency in the fluorescence microscopy analysis. Moreover, QD-Cramoll conjugates remained brightly fluorescent and preserved identical biological activity according to hemagglutination assays. Flow cytometry revealed that approximately 17% of C. albicans cells were labeled after incubation with covalent conjugates, while approximately 92% of cells were labeled by adsorption conjugates (both at pH7.0). Inhibition assays confirmed QD-Cramoll specificity, which reduced the labeling to at most 3%. Therefore, the conjugates obtained by adsorption (pH7.0) proved to be promising and versatile fluorescent tools for glycobiology.

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs.

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model's ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector

Cloning of multiple genes in a single vector has greatly facilitated both basic and translational studies that require co-expression of multiple factors or multi-units of complex protein. Many strategies have been adopted, among which 2A “self-cleaving” peptides have garnered increased interest for their polycistronic nature, small size and high “cleavage” efficiency. However, broad application of 2 A peptides is limited by the lack of systematic comparison of different 2As alone or in combination. Here we characterized the effect of varying gene position and 2As on the expression of proteins encoded in bi-, tri-, or quad-cistronic constructs. Using direct cardiac reprogramming as an example, we further determined the effect of varied 2As on the efficiency of fluorescent cell labeling and cell fate conversion. We found that the expression of fluorophores decreased as it was moved towards the end of the construct while reprogramming was most efficient with the fluorophore at the second position. Moreover, quad-cistronic TPE2A constructs resulted in more efficient reprogramming than 3P2A or PTE2A constructs. We expect that the bi-, tri-, and quad-cistronic vectors constructed here and our results on protein expression ratios from different 2A constructs could serve to guide future utilization of 2A peptides in basic research and clinical applications.

Abstract A25: High-resolution visualization of immune response across the intact melanoma tumor

Abstract Tumor immunotherapy relies on improved anti-tumor activity of the immune cells. Efficacious immunotherapy often correlates with increased CD8+ T cell infiltration within the tumor, subsequently resulting into reduction in tumor growth and morphological remodeling. However, pan- melanoma visualization of immune cell infiltration and associated macro-scale alterations in tumor morphology and vasculature remains extremely challenging. This is largely owing to the dense pigmentation, thick extracellular matrix deposition and friable texture of the tumor, rendering it optically inaccessible using the routine histology and organ clarification techniques. Here, we describe a rapid and inexpensive optical clarification technique that enables examination of immune infiltration as well as vascular changes, both at tissue level as well as single cell-resolution within an intact tumor. The aforementioned technique, termed Tumor-optimized three-dimensional imaging of solvent cleared organs (T-DISCO), combines in vivo fluorescent labeling of the endogenous immune cells with organic solvents-based tissue clarification and eventual image acquisition using a standard confocal microscope. This technique when applied to the B16F10 melanoma model undergoing a multi-component immunotherapy, revealed changes within the tumor microenvironment, including vascular normalization and intra-tumoral trafficking of the T cells. Thus, T-DISCO represents a powerful tool to examine and investigate immune cell behavior deep within the intact tumor. Citation Format: Sudha Kumari, Kelly Moynihan, Darrell J. Irvine. High-resolution visualization of immune response across the intact melanoma tumor. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A25.

Technetium(I) Complexes of Bathophenanthrolinedisulfonic Acid.

Bathophenanthrolinedisulfonate (BPS) complexes of technetium(I) of the type [Tc(CO)3(BPS)(L)]n (L = imidazole derivatives) were synthesized and evaluated both in vitro and in vivo. [99mTc(CO)3(BPS)(MeIm)]- (MeIm = 1-methyl-1H-imidazole) was prepared in near-quantitative yield using a convenient two-step, one-pot labeling procedure. A targeted analogue capable of binding regions of calcium turnover associated with bone metabolism was also prepared. Here, a bisphosphonate was linked to the metal through an imidazole ligand to give [99mTc(CO)3(BPS)(ImAln)]2- (ImAln = an imidazole-alendronate ligand) in high yield. The technetium(I) complexes were stable in vitro, and in biodistribution studies, [99mTc(CO)3(BPS)(ImAln)]2- exhibited rapid clearance from nontarget tissues and significant accumulation in the shoulder (7.9 ± 0.2% ID/g) and knees (15.1 ± 0.9% ID/g) by 6 h, with the residence time in the skeleton reaching 24 h. A rhenium analogue, which is luminescent and has the same structure, was also prepared and used for fluorescence labeling of cells in vitro. The data reported demonstrate the potential of this class of compounds for use in creating isostructural optical and nuclear probes.