Labeling Of Cells Research Articles

FNDs for the selective labelling and localized free radical measurements in sperm cells

FNDs for the selective labelling and localized free radical measurements in sperm cells

Direct labeling of milk cells without centrifugation for counting total and differential somatic cells using flow cytometry.

Somatic cell count (SCC) in milk is an essential indicator for defining and managing udder health. However, analyzing differential SCC (dSCC) can be helpful in determining the type or evolution stage of mastitis. A high abundance of polymorphonuclear cells (PMN) is associated with acute mastitis; however, the status of a chronic disease is less well characterized. A method capable of analyzing SCC and dSCC can prove to be a helpful tool for monitoring the status of evolution of mastitis disease in a better way. Therefore, a new direct-flow cytometry method was developed to count and differentiate somatic cells in milk without the steps of centrifugation or washing, avoiding variabilities that occur due to enrichment or loss of specific cell types. In this new method, SCC is analyzed using the method of DNA staining with Hoechst stain, whereas dSCC are analyzed using specific antibodies targeting 2 main cell types associated with mastitis: PMN cells and antigen-presenting cells, which are associated with innate and adaptive immunity. Equivalent SCC values were obtained between the new method and the routine ISO 13366-2 method in a comparison of 240 raw milk samples. Furthermore, dSCC results were confirmed by microscopy after May-Gründwald-Giemsa staining in 165 quarter milk samples from healthy and diseased cows. The method was verified with fluorescence microscopy on the 2 targeted cell types and in raw milk samples. The newly developed method is independent of any instrument and can be further designed to differentiate other cell types and animal species by selecting appropriate antibodies.

The Use of 99mTc-Labeled Stem Cells for Evaluation of Chronic Kidney Disease.

The labeling of stem cells with radionuclides allows in vivo monitoring of cell migration and homing. Here, we describe the labeling of mononuclear stem cells with 99mTc and show their biodistribution in preclinical models and patients with chronic kidney disease.

Long-term and non-invasive in vivo tracking of DiD dye-labeled human hepatic progenitors in chronic liver disease models.

Chronic liver diseases (CLD) are the major public health burden due to the continuous increasing rate of global morbidity and mortality. The inherent limitations of organ transplantation have led to the development of stem cell-based therapy as a supportive and promising therapeutic option. However, identifying the fate of transplanted cells in vivo represents a crucial obstacle. To evaluate the potential applicability of DiD dye as a cell labeling agent for long-term, and non-invasive in vivo tracking of transplanted cells in the liver. Magnetically sorted, epithelial cell adhesion molecule positive (1 × 106 cells/mL) fetal hepatic progenitor cells were labeled with DiD dye and transplanted into the livers of CLD-severe combined immunodeficiency (SCID) mice. Near-infrared (NIR) imaging was performed for in vivo tracking of the DiD-labeled transplanted cells along with colocalization of hepatic markers for up to 80 d. The existence of human cells within mouse livers was identified using Alu polymerase chain reaction and sequencing. NIR fluorescence imaging of CLD-SCID mice showed a positive fluorescence signal of DiD at days 7, 15, 30, 45, 60, and 80 post-transplantation. Furthermore, positive staining of cytokeratin, c-Met, and albumin colocalizing with DiD fluorescence clearly demonstrated that the fluorescent signal of hepatic markers emerged from the DiD-labeled transplanted cells. Recovery of liver function was also observed with serum levels of glutamic-oxaloacetic transaminase, glutamate-pyruvate transaminase, and bilirubin. The detection of human-specific Alu sequence from the transplanted mouse livers provided evidence for the survival of transplanted cells at day 80. DiD-labeling is promising for long-term and non-invasive in vivo cell tracking, and understanding the regenerative mechanisms incurred by the transplanted cells.

Nestin is a marker of unipotent embryonic and adult progenitors differentiating into an epithelial cell lineage of the hair follicles

Nestin is an intermediate filament protein transiently expressed in neural stem/progenitor cells. We previously demonstrated that outer root sheath (ORS) keratinocytes of adult hair follicles (HFs) in mice descend from nestin-expressing cells, despite being an epithelial cell lineage. This study determined the exact stage when nestin-expressing ORS stem/precursor cells or their descendants appear during HF morphogenesis, and whether they are present in adult HFs. Using Nes-Cre/CAG-CAT-EGFP mice, in which enhanced green fluorescent protein (EGFP) is expressed following Cre-based recombination driven by the nestin promoter, we found that EGFP+ cells appeared in the epithelial layer of embryonic HFs as early as the peg stage. EGFP+ cells in hair pegs were positive for keratin 14 (K14) and K5, but not vimentin, SOX2, SOX10, or S100 alpha 6. Tracing of tamoxifen-induced EGFP+ cells in postnatal Nes-CreERT2/CAG-CAT-EGFP mice revealed labeling of some isthmus HF epithelial cells in the first anagen stage. EGFP+ cells in adult HFs were not immunolabeled for K15, an HF multipotent stem cell marker. However, when hairs were depilated in Nes-CreERT2/CAG-CAT-EGFP mice to induce the anagen stage after tamoxifen injection, the majority of ORS keratinocytes in depilation-induced anagen HFs were labeled for EGFP. Our findings indicate that nestin-expressing unipotent progenitor cells capable of differentiating into ORS keratinocytes are present in HF primordia and adult HFs.

Applying a Knock-In Strategy to Create Reporter-Tagged Knockout Alleles in Axolotls.

Tetrapod species axolotls exhibit the powerful capacity to fully regenerate their tail and limbs upon injury, hence serving as an excellent model organism in regenerative biology research. Developing proper molecular and genetic tools in axolotls is an absolute necessity for deep dissection of tissue regeneration mechanisms. Previously, CRISPR-/Cas9-based knockout and targeted gene knock-in approaches have been established in axolotls, allowing genetically deciphering gene function, labeling, and tracing particular types of cells. Here, we further extend the CRISPR/Cas9 technology application and describe a method to create reporter-tagged knockout allele in axolotls. This method combines gene knockout and knock-in and achieves loss of function of a given gene and simultaneous labeling of cells expressing this particular gene, that allows identification, tracking of the "knocking out" cells. Our method offers a useful gene function analysis tool to the field of axolotl developmental and regenerative research.

SpotitPy: a semi-automated tool for object-based co-localization of fluorescent labels in microscopy images

BackgroundIn fluorescence microscopy, co-localization refers to the spatial overlap between different fluorescent labels in cells. The degree of overlap between two or more channels in a microscope may reveal a physical interaction or topological functional interconnection between molecules. Recent advances in the imaging field require the development of specialized computational analysis software for the unbiased assessment of fluorescently labelled microscopy images.ResultsHere we present SpotitPy, a semi-automated image analysis tool for 2D object-based co-localization. SpotitPy allows the user to select fluorescent labels and perform a semi-automated and robust segmentation of the region of interest in distinct cell types. The workflow integrates advanced pre-processing manipulations for de-noising and in-depth semi-automated quantification of the co-localized fluorescent labels in two different channels. We validated SpotitPy by quantitatively assessing the presence of cytoplasmic ribonucleoprotein granules, e.g. processing (P) bodies, under conditions that challenge mRNA translation, thus highlighting SpotitPy benefits for semi-automatic, accurate analysis of large image datasets in eukaryotic cells. SpotitPy comes in a command line interface or a simple graphical user interphase and can be used as a standalone application.ConclusionsOverall, we present a novel and user-friendly tool that performs a semi-automated image analysis for 2D object-based co-localization. SpotitPy can provide reproducible and robust quantifications for large datasets within a limited timeframe. The software is open-source and can be found in the GitHub project repository: (https://github.com/alexiaales/SpotitPy).

Evaluation of a library of loxP variants with a wide range of recombination efficiencies by Cre.

Sparse labeling of individual cells is an important approach in neuroscience and many other fields of research. Various methods have been developed to sparsely label only a small population of cells; however, there is no simple and reproducible strategy for managing the probability of sparse labeling at desired levels. Here, we aimed to develop a novel methodology based on the Cre-lox system to regulate sparseness at desired levels, and we purely analyzed cleavage efficiencies of loxP mutants by Cre. We hypothesized that mutations in the loxP sequence reduce the recognition efficiency by Cre, which enables the regulation of the sparseness level of gene expression. In this research, we mutagenized the loxP sequence and analyzed a library of loxP variants. We evaluated more than 1000 mutant loxP sequences, including mutants with reduced excision efficiencies by Cre ranging from 0.51% to 59%. This result suggests that these mutant loxP sequences can be useful in regulating the sparseness of genetic labeling at desired levels.

Development of Red-Emissive Porphyrin Graphene Quantum Dots (PGQDs) for Biological Cell-Labeling Applications.

Red and near-infrared emission is a highly desirable feature for fluorescent nanoparticles in biological applications mainly due to longer wavelengths more easily being able to deeply penetrate tissues, organs, skin, and other organic components, while less autofluorescence interference would be produced. Additionally, graphene quantum dots (GQDs) that contain unique optical and electrical features have been targeted for their use in cell labeling applications as well as environmental analysis. Their most desirable features come in the form of low toxicity and biocompatibility; however, GQDs are frequently reported to have blue or green emission light and not the more advantageous red/NIR emission light. Furthermore, porphyrins are a subgroup of heterocyclic macrocycle organic compounds that are also naturally occurring pigments in nature that already contain the desired red-emission fluorescence. Therefore, porphyrins have been used previously to synthesize nanomaterials and for nanoparticle doping in order to incorporate the red/NIR emission light property into particles that otherwise do not contain the desired emission light. Meso-tetra(4-carboxyphenyl)porphine (TCPP) is one type of porphyrin with a large conjugated π-electron system and four carboxyl groups on its exterior benzene rings. These two key characteristics of TCPP make it ideal for incorporation into GQDs, as it would design and synthesize red-emissive material as well as give rise to excellent water solubility. In this work, TCPP is used in tangent with cis-cyclobutane-1,2-dicarboxylic acid (CBDA-2), a biomass derived organic molecule, to synthesize "green" porphyrin-based graphene quantum dots (PGQDs) with red-emission. The obtained PGQDs were characterized by various analytical methods. Utilizing TEM, HRTEM, and DLS the size distribution of the particles was determined to be 7.9 ± 4.1, well within the quantum dot range of 2-10 nm. FT-IR, XPS, and XRD depicted carbon, nitrogen, and oxygen as the main elemental components with carbon being in the form of graphene and the main porphyrin ring of TCPP remaining present in the final PGQDs product. Lastly, absorption and fluorescence spectroscopy determined the excitation wavelength at 420 nm and the emission at 650 nm which was successfully utilized in the imaging of HeLa cells using confocal microscopy.

Tetraplex Immunophenotyping of Cell Surface Proteomes via Synthesized Plasmonic Nanotags and Portable Raman Spectroscopy.

Multiplex immunophenotyping of cell surface proteomes is useful for cell characterization as well as providing valuable information on a patient's physiological or pathological state. Current methods for multiplex immunophenotyping of cell surface proteomes still have associated technical pitfalls in terms of limited multiplexing capability, challenging result interpretation, and large equipment footprint limited to use in a laboratory setting. Herein, we presented a portable surface-enhanced Raman spectroscopy (SERS) assay for multiplex cell surface immunophenotyping. We synthesized and functionalized customizable SERS nanotags for cell labeling and subsequent signal measurement using a portable Raman spectrometer. We extensively evaluated and validated the analytical assay performance of the portable SERS immunophenotyping assay in two different cellular models (red blood cells and breast cancer cells). In terms of analytical specificity, the cell surface immunophenotyping of both red blood cells and breast cancer cells correlated well with flow cytometry. The portable SERS immunophenotyping assay also has comparable analytical repeatability to flow cytometry, with coefficient of variation values of 21.89-23.33% and 6.88-17.32% for detecting red blood cells and breast cancer cells, respectively. The analytical detection limits were 77 cells/mL for red blood cells and 1-17 cells/mL for breast cancer cells. As an alternative to flow cytometry, the portable SERS immunophenotyping assay demonstrated excellent analytical assay performance and possessed advantages such as quick sample-to-result turnaround time, multiplexing capability, and small equipment footprint.

Optimization of superfluorinated PLGA nanoparticles for enhanced cell labelling and detection by 19F-MRI

Fluorine-19 (19F) Magnetic Resonance Imaging (MRI) is an emergent imaging technique for molecular imaging and cell tracking. Lack of intrinsic 19F signals in tissues allows unambiguous in vivo detection of exogenous fluorinated probes, complementary to the anatomical and multiparametric information obtained by standard 1H-MRI. However, the intrinsic low sensitivity of MRI technique requires the need of designing increasingly effective fluorinated tracers. PERFECTA, with its 36 magnetically equivalent 19F atoms and a designed branched molecular structure, represents an excellent superfluorinated tracer. In this paper, we report the development of PERFECTA loaded PLGA NPs stabilized by different coatings as promising 19F-MRI probes. The results clearly show the optimal cellular uptake of the produced colloidally stable PERFECTA loaded PLGA NPs without impact on cells viability. Importantly, NPs stabilization with the anionic surfactant sodium cholate (NaC) clearly enhances NPs internalization within cells with respect to PVA-coated NPs. Moreover, the optimized NPs are characterized by shorter T1 relaxation times with respect to other PERFECTA formulations that would allow the increase of 19F-MRI sensitivity with fast imaging acquisitions.

Myoglobin regulates fatty acid trafficking and lipid metabolism in mammary epithelial cells.

Myoglobin (MB) is known to bind and deliver oxygen in striated muscles at high expression levels. MB is also expressed at much reduced levels in mammary epithelial cells, where the protein´s function is unclear. In this study, we aim to determine whether MB impacts fatty acid trafficking and facilitates aerobic fatty acid ß-oxidation in mammary epithelial cells. We utilized MB-wildtype versus MB-knockout mice and human breast cancer cells to examine the impact of MB and its oxygenation status on fatty acid metabolism in mouse milk and mammary epithelia. MB deficient cells were generated through CRISPR/Cas9 and TALEN approaches and exposed to various oxygen tensions. Fatty acid profiling of milk and cell extracts were performed along with cell labelling and immunocytochemistry. Our findings show that MB expression in mammary epithelial cells promoted fatty acid oxidation while reducing stearyl-CoA desaturase activity for lipogenesis. In cells and milk product, presence of oxygenated MB significantly elevated indices of limited fatty acid ß-oxidation, i.e., the organelle-bound removal of a C2 moiety from long-chain saturated or monounsaturated fatty acids, thus shifting the composition toward more saturated and shorter fatty acid species. Presence of the globin also increased cytoplasmic fatty acid solubility under normoxia and fatty acid deposition to lipid droplets under severe hypoxia. We conclude that MB can function in mammary epithelia as intracellular O2-dependent shuttle of oxidizable fatty acid substrates. MB’s impact on limited oxidation of fatty acids could generate inflammatory mediator lipokines, such as 7-hexadecenoate. Thus, the novel functions of MB in breast epithelia described herein range from controlling fatty acid turnover and homeostasis to influencing inflammatory signalling cascade. Future work is needed to analyse to what extent these novel roles of MB also apply to myocytic cell physiology and malignant cell behaviour, respectively.

Bio-Nanohybrid Gelatin/Quantum Dots for Cellular Imaging and Biosensing Applications.

The bio-nanohybrid gelatin protein/cadmium sulfide (Gel/CdS) quantum dots (QDs) have been designed via a facile one-pot strategy. The amino acids group of gelatin chelate Cd2+ and grow CdS QDs without any agglomeration. The 1H NMR spectra indicate that during the above process there are no alterations of the gelatin protein structure conformation and chemical functionalities. The prepared Gel/CdS QDs were characterized and their potential as a system for cellular imaging and the electrochemical sensor for hydrogen peroxide (H2O2) detection applications were investigated. The obtained results demonstrate that the developed Gel/CdS QDs system could offer a simple and convenient operating strategy both for the class of contrast agents for cell labeling and electrochemical sensors purposes.

CellDestiny: A RShiny application for the visualization and analysis of single-cell lineage tracing data.

Single-cell lineage tracing permits the labeling of individual cells with a heritable marker to follow the fate of each cell’s progeny. Over the last twenty years, several single-cell lineage tracing methods have emerged, enabling major discoveries in developmental biology, oncology and gene therapies. Analytical tools are needed to draw meaningful conclusions from lineage tracing measurements, which are characterized by high variability, sparsity and technical noise. However, the single cell lineage tracing field lacks versatile and easy-to-use tools for standardized and reproducible analyses, in particular tools accessible to biologists. Here we present CellDestiny, a RShiny app and associated web application developed for experimentalists without coding skills to perform visualization and analysis of single cell lineage-tracing datasets through a graphical user interface. We demonstrate the functionality of CellDestiny through the analysis of (i) lentiviral barcoding datasets of murine hematopoietic progenitors; (ii) published integration site data from Wiskott-Aldrich Symdrome patients undergoing gene-therapy treatment; and (iii) simultaneous barcoding and transcriptomic analysis of murine hematopoietic progenitor differentiation in vitro. In summary, CellDestiny is an easy-to-use and versatile toolkit that enables biologists to visualize and analyze single-cell lineage tracing data.

Fluorine labelling of therapeutic human tolerogenic dendritic cells for 19F-magnetic resonance imaging.

Tolerogenic dendritic cell (tolDC) therapies aim to restore self-tolerance in patients suffering from autoimmune diseases. Phase 1 clinical trials with tolDC have shown the feasibility and safety of this approach, but have also highlighted a lack of understanding of their distribution in vivo. Fluorine-19 magnetic resonance imaging (19F-MRI) promises an attractive cell tracking method because it allows for detection of 19F-labelled cells in a non-invasive and longitudinal manner. Here, we tested the suitability of nanoparticles containing 19F (19F-NP) for labelling of therapeutic human tolDC for detection by 19F-MRI. We found that tolDC readily endocytosed 19F-NP with acceptable effects on cell viability and yield. The MRI signal-to-noise ratios obtained are more than sufficient for detection of the administered tolDC dose (10 million cells) at the injection site in vivo, depending on the tissue depth and the rate of cell dispersal. Importantly, 19F-NP labelling did not revert tolDC into immunogenic DC, as confirmed by their low expression of typical mature DC surface markers (CD83, CD86), low secretion of pro-inflammatory IL-12p70, and low capacity to induce IFN-γ in allogeneic CD4+ T cells. In addition, the capacity of tolDC to secrete anti-inflammatory IL-10 was not diminished by 19F-NP labelling. We conclude that 19F-NP is a suitable imaging agent for tolDC. With currently available technologies, this imaging approach does not yet approach the sensitivity required to detect small numbers of migrating cells, but could have important utility for determining the accuracy of injecting tolDC into the desired target tissue and their efflux rate.

Switching of Photocatalytic Tyrosine/Histidine Labeling and Application to Photocatalytic Proximity Labeling.

Weak and transient protein interactions are involved in dynamic biological responses and are an important research subject; however, methods to elucidate such interactions are lacking. Proximity labeling is a promising technique for labeling transient ligand–binding proteins and protein–protein interaction partners of analytes via an irreversible covalent bond. Expanding chemical tools for proximity labeling is required to analyze the interactome. We developed several photocatalytic proximity-labeling reactions mediated by two different mechanisms. We found that numerous dye molecules can function as catalysts for protein labeling. We also identified catalysts that selectively modify tyrosine and histidine residues and evaluated their mechanisms. Model experiments using HaloTag were performed to demonstrate photocatalytic proximity labeling. We found that both ATTO465, which catalyzes labeling by a single electron transfer, and BODIPY, which catalyzes labeling by singlet oxygen, catalyze proximity labeling in cells.

Developing Photoaffinity Probes for Dopamine Receptor D2 to Determine Targets of Parkinson's Disease Drugs.

Dopaminergic pathways control highly consequential aspects of physiology and behavior. One of the most therapeutically important and best-studied receptors in these pathways is dopamine receptor D2 (DRD2). Unfortunately, DRD2 is challenging to study with traditional molecular biological techniques, and most drugs designed to target DRD2 are ligands for many other receptors. Here, we developed probes able to both covalently bind to DRD2 using photoaffinity labeling and provide a chemical handle for detection or affinity purification. These probes behaved like good DRD2 agonists in traditional biochemical assays and were able to perform in chemical–biological assays of cell and receptor labeling. Rat whole brain labeling and affinity enrichment using the probes permitted proteomic analysis of the probes’ interacting proteins. Bioinformatic study of the hits revealed that the probes bound noncanonically targeted proteins in Parkinson’s disease network as well as the retrograde endocannabinoid signaling, neuronal nitric oxide synthase, muscarinic acetylcholine receptor M1, GABA receptor, and dopamine receptor D1 (DRD1) signaling networks. Follow-up analysis may yield insights into how this pathway relates specifically to Parkinson’s disease symptoms or provide new targets for treatments. This work reinforces the notion that the combination of chemical biology and omics-based approaches provides a broad picture of a molecule’s “interactome” and may also give insight into the pleiotropy of effects observed for a drug or perhaps indicate new applications.

Artificial light-harvesting systems and their applications in photocatalysis and cell labeling

• The artificial light-harvesting systems based on non-covalent interactions are discussed. • The artificial light-harvesting systems with two-step sequential energy transfer are discussed. • The applications of artificial light-harvesting systems in photocatalysis and cell labeling are discussed. Photosynthesis is the basis for the survival of organisms in nature; consequently, the fabrication of artificial light-harvesting systems (LHSs) that simulate natural photosynthesis is of significant interest. Recently, a variety of artificial LHSs have been successfully constructed using fluorescence resonance energy transfer (FRET). However, it is crucial to fabricate artificial LHSs with a sequential energy transfer process when considering that the natural photosynthetic process involves a multistep sequential energy transfer process rather than a simple one-step energy transfer. Moreover, many previously reported LHSs have been used as imaging agents for cell labeling and bioimaging or as catalysts in photocatalytic reactions, showing promise for applications simulating natural photosynthesis. In this review, we have summarized recently published representative work on artificial LHSs. In addition, the application of LHSs in photocatalysis and cell labeling has been described in detail.

In vivo ultrasound imaging of macrophages using acoustic vaporization of internalized superheated nanodroplets

Ultrasound (US) not only detects but also interacts with its contrast agents, potentially allowing the use of perfluorocarbon-filled nanodroplets (NDs) or microbubbles (MBs) for both the detection and treatment of deep-seated tumors. Cells labeled using MBs to allow their detection in vivo with clinical ultrasound scanner presents several advantages over PET and MRI: Ultrasound is 2–3 orders of magnitude more sensitive and can detect a single acoustically labeled cell in vivo. Furthermore, with high ultrasound intensities, drugs or genes that are loaded on NDs or MBs can be delivered with spatiotemporal control. Although the acoustic labeling of stem cells for in vivo imaging using MBs has been reported, the ability to load and image internalized low boiling point NDs has not been explored yet. We used perfluorobutane NDs to acoustically label macrophages, which were viable following post internalization and ultrasound-mediated phase change. We documented that internalized NDs are stable for at least 8 h, which is needed for macrophages to accumulate to diseased sites. Labeled macrophage accumulated in the liver of healthy rats as stable intracellular NDs were vaporized into MBs using a clinical scanner (Siemens ACUSON Sequoia, 10L4 transducer at 4 MHz, 1.4 MI) and remained visible after vaporization.

A Peptide-Based Ligand-Directed Chemistry Enables Protein Functionalization.

The ligand-directed (LD) chemistry provides powerful tools for site-specific modification of proteins. We utilized a peptide with an appended methionine (Met) as a ligand; then, the Met thioether was modified into sulfonium which enabled a proximity induced group transfer onto protein cysteine in the vicinity upon peptide-target binding. The sulfonium warhead could be easily constructed with unprotected peptides, and the transferable group scope was conducted on model protein PDZ and its ligand peptides. In addition, a living cell labeling was successfully achieved.