Fluorescence Labeling Of Cells Research Articles

Poly(N,N-dimethylacrylamide)-coated upconverting NaYF4:Yb,Er@NaYF4:Nd core\u2013shell nanoparticles for fluorescent labeling of carcinoma cells

Upconverting luminescent lanthanide-doped nanoparticles (UCNP) belong to promising new materials that absorb infrared light able to penetrate in the deep tissue level, while emitting photons in the visible or ultraviolet region, which makes them favorable for bioimaging and cell labeling. Here, we have prepared upconverting NaYF4:Yb,Er@NaYF4:Nd core–shell nanoparticles, which were coated with copolymers of N,N-dimethylacrylamide (DMA) and 2-(acryloylamino)-2-methylpropane-1-sulfonic acid (AMPS) or tert-butyl [2-(acryloylamino)ethyl]carbamate (AEC-Boc) with negative or positive charges, respectively. The copolymers were synthesized by a reversible addition-fragmentation chain transfer (RAFT) polymerization, reaching Mn ~ 11 kDa and containing ~ 5 mol% of reactive groups. All copolymers contained bisphosphonate end-groups to be firmly anchored on the surface of NaYF4:Yb,Er@NaYF4:Nd core–shell nanoparticles. To compare properties of polymer coatings, poly(ethylene glycol)-coated and neat UCNP were used as a control. UCNP with various charges were then studied as labels of carcinoma cells, including human hepatocellular carcinoma HepG2, human cervical cancer HeLa, and rat insulinoma INS-1E cells. All the particles proved to be biocompatible (nontoxic); depending on their ξ-potential, the ability to penetrate the cells differed. This ability together with the upconversion luminescence are basic prerequisites for application of particles in photodynamic therapy (PDT) of various tumors, where emission of nanoparticles in visible light range at ~ 650 nm excites photosensitizer.

Abstract 3055: Clonogenic high-throughput assay for screening radiation modulators

Abstract We present here the development of a high-throughput clonogenic methodology for the identification of novel radiation modulators, with critical and laborious steps automated for speed and ease of use. Clonogenic assays have long been the benchmark methodology for assessing reproductive cell death following treatment with ionizing radiation, alone or in combination with a variety of chemotherapeutic agents (cytotoxic, genotoxic, targeted therapies). However, owning to the large growth formats and intrinsic laborious nature of clonogenic assays heretofore large-scale screening for radiation modulators with this assay has been impractical. Further, presently available large-scale cellular growth screens (e.g. MTT type assays) have proven unreliable at reproducibly identifying clinically relevant radiation modulators. As a result, numerous attempts have been made at developing a streamlined clonogenic process, with modest degrees of success. Here we present the development of an automated miniaturized clonogenic methodology that finally provides for the high throughput screening of novel radiation modulators in cell lines representing seven cancer indications where radiation therapy is the standard of care (Esophageal cancer, Glioblastoma, Head and Neck cancer, metastatic Melanoma, Non-small-cell lung carcinoma, Rectal cancer, and Soft tissue sarcoma). First, we have identified multiple cell lines for each cancer indication that form consistently small and discrete colonies, making them amenable to clonogenic growth in 24- and/or 96-well formats. Second, we have identified conditions in which chemoradiation response is identical between traditional and miniaturized clonogenic formats. Third, we have developed fluorescent cell labeling protocols that allow for live cell colony counting, streamlining the assay and allowing for real-time monitoring over the course of each experiment. Fourth, we have developed software for the automated calling/quantification of clonogenic images, calculation of α/β ratio, dose modifying factor, and calling of hits in large scale screening. Fifth, we have developed an instrument that in large scale is capable of robotic liquid handling, irradiation, drug treatment, and incubation of clonogenic plates in an integrated manner. Preliminary screens with this methodology have demonstrated its ability to accurately identify known radiation modulators. Taken together these new technologies provide a platform to allow for the high-throughput clonogenic screening of drug libraries to identify novel radiation modulators. Further, this methodology is adaptable for identification of novel drug-drug combinations using a clonogenic endpoint. This work is funded in part by NCI SBIR Phase II contract No. 75N9109C0038. Citation Format: Barbara Frederick, Nathan Gomes, Tin Tin Su. Clonogenic high-throughput assay for screening radiation modulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3055.

Fluorescent labeling of the root cap cells with the bioactive NBD-S chemical probe based on the cellulose biosynthesis inhibition herbicides

Development of the methods to examine the molecular targets of biologically active compounds is one of the most important subjects in experimental biology/biochemistry. To evaluate the usability of the (7-nitro-2,1,3-benzoxadiazole)-thioether (NBD-S) probe for this purpose, bioactive chemical probe (1) as the cellulose biosynthesis (CB) inhibitor was synthesized and tested. As a result, a variety of fluorescently-labeled particles and organelles were found in the columella root cap cells of radish plants. Of note, well-defined cellular organelles were clearly recognized in the detaching root cap cells (border-like cells). These results imply that the bioactive NBD-S chemical probe could be a valuable direct-labeling reagent. Analysis of these fluorescent substances would be helpful in providing new information on defined molecular targets and events.

Abstract PO-080: Clonogenic high-throughput assay for screening radiation modulators

Abstract We present here the development of a high-throughput clonogenic methodology for the identification of novel radiation modulators, with critical and laborious steps automated for speed and ease of use. Clonogenic assays have long been the benchmark methodology for assessing reproductive cell death following treatment with ionizing radiation, alone or in combination with a variety of chemotherapeutic agents (cytotoxic, genotoxic, targeted therapies). However, owning to the large growth formats and intrinsic laborious nature of clonogenic assays heretofore large-scale screening for radiation modulators with this assay has been impractical. Further, presently available large-scale cellular growth screens (e.g. MTT type assays) have proven unreliable at reproducibly identifying clinically relevant radiation modulators. As a result, numerous attempts have been made at developing a streamlined clonogenic process, with modest degrees of success. Here we present the development of an automated miniaturized clonogenic methodology that finally provides for the high throughput screening of novel radiation modulators in cell lines representing seven cancer indications where radiation therapy is the standard of care (Esophageal cancer, Glioblastoma, Head and Neck cancer, metastatic Melanoma, Non-small-cell lung carcinoma, Rectal cancer, and Soft tissue sarcoma). First, we have identified multiple cell lines for each cancer indication that form consistently small and discrete colonies, making them amenable to clonogenic growth in 24- and/or 96-well formats. Second, we have identified conditions in which chemoradiation response is identical between traditional and miniaturized clonogenic formats. Third, we have developed fluorescent cell labeling protocols that allow for live cell colony counting, streamlining the assay and allowing for real-time monitoring over the course of each experiment. Fourth, we have developed software for the automated calling/quantification of clonogenic images, calculation of α/β ratio, dose modifying factor, and calling of hits in large scale screening. Fifth, we have developed an instrument that in large scale is capable of robotic liquid handling, irradiation, drug treatment, and incubation of clonogenic plates in an integrated manner. Preliminary screens with this methodology have demonstrated its ability to accurately identify known radiation modulators. Taken together these new technologies provide a platform to allow for the high-throughput clonogenic screening of drug libraries to identify novel radiation modulators. Further, this methodology is adaptable for identification of novel drug-drug combinations using a clonogenic endpoint. This work is funded in part by NCI SBIR Phase II contract No. 75N9109C0038. Citation Format: Barbara Frederick, Nathan Gomes, Tin Tin Su. Clonogenic high-throughput assay for screening radiation modulators [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-080.

In vivo analysis of noise dependent activation of white blood cells and microvascular dysfunction in mice

This article contains supporting information on data collection for the research article entitled “Aircraft noise exposure drives the activation of white blood cells and induces microvascular dysfunction in mice” by Eckrich et al. We found that noise-induced stress triggered microvascular dysfunction via involvement of innate immune-derived reactive oxygen species. In this article, we present the instrumentation of mice with dorsal skinfold chambers for in vivo microscopic imaging of blood flow, interaction of leukocytes with the vascular wall (also by fluorescent labelling of blood cells) and vessel diameter. In addition, we explain the preparation of cerebral arterioles for measurement of vascular reactivity in vitro.•visualization of noise-dependent effects in dorsal skinfold chamber.•in vivo microscopy of noise-dependent activation of white blood cells.•analysis of noise-dependent microvascular dysfunction in dorsal skinfold chamber and cannulated cerebral arterioles.

Application of Safirinium N-Hydroxysuccinimide Esters to Derivatization of Peptides for High-Resolution Mass Spectrometry, Tandem Mass Spectrometry, and Fluorescent Labeling of Bacterial Cells.

Mass spectrometry methods are commonly used in the identification of peptides and biomarkers. Due to a relatively low abundance of proteins in biological samples, there is a need for the development of novel derivatization methods that would improve MS detection limits. Hence, novel fluorescent N–hydroxysuccinimide esters of dihydro-[1,2,4]triazolo[4,3-a]pyridin-2-ium carboxylates (Safirinium P dyes) have been synthesized. The obtained compounds, which incorporate quaternary ammonium salt moieties, easily react with aliphatic amine groups of peptides, both in solution and on the solid support; thus, they can be applied for derivatization as ionization enhancers. Safirinium tagging experiments with ubiquitin hydrolysate revealed that the sequence coverage level was high (ca. 80%), and intensities of signals were enhanced up to 8-fold, which proves the applicability of the proposed tags in the bottom–up approach. The obtained results confirmed that the novel compounds enable the detection of trace amounts of peptides, and fixed positive charge within the tags results in high ionization efficiency. Moreover, Safirinium NHS esters have been utilized as imaging agents for fluorescent labeling and the microscopic visualization of living cells such as E. coli Top10 bacterial strain.

Tumor-cell detection, labeling and phenotyping with an electron-doped bifunctional signal-amplifier

Cancer cell enumeration and phenotyping can predict the prognosis and the therapy efficacy in patients, yet it remains challenging to detect the rare tumor cells. Herein, we report an octopus-inspired, bifunctional aptamer signal amplifier-based cytosensor (OApt-cytosensor) for sensitive cell analysis. By assembling high-affinity antibodies on an electrode surface, the target cells could be specifically captured and thus been sandwiched by the cell surface marker-specific DNA aptamers. These on-cell aptamers function as electrochemical signal amplifiers by base-selective electronic doping with methylene blue. Such a sandwich configuration enables highly sensitive cell detection down to 10 cells/mL (equal to ~1–2 cells at a sampling volume of 150 μL), even in a large excess of nontarget blood cells. This approach also reveals the cell-surface markers and tracks the cellular epithelial-to-mesenchymal transition induced by signaling regulators. Furthermore, the electron-doped aptamer shows remarkable cell fluorescent labeling that guides the release of the captured cells from electrode surface via electrochemistry. These features make OApt-cytosensor a promising tool in revealing the heterogeneous cancer cells and anticancer drug screening at the single-cell level.

CAFs and Cancer Cells Co-Migration in 3D Spheroid Invasion Assay.

In many solid tumors, collective cell invasion prevails over single-cell dissemination strategies. Collective modes of invasion often display specific front/rear cellular organization, where invasive leader cells arise from cancer cell populations or the tumor stroma. Collective invasion involves coordinated cellular movements which require tight mechanical crosstalk through specific combinations of cell-cell interactions and cell-matrix adhesions.Cancer Associated Fibroblasts (CAFs) have been recently reported to drive the dissemination of epithelial cancer cells through ECM remodeling and direct intercellular contact. However, the cooperation between tumor and stromal cells remains poorly understood. Here we present a simple spheroid invasion assay to assess the role of CAFs in the collective migration of epithelial tumor cells. This method enables the characterization of 3D spheroid invasion patterns through live cell fluorescent labeling combined with spinning disc microscopy. When embedded in extracellular matrix, the invasive strands of spheroids can be tracked and leader/follower organization of CAFs and cancer cells can be quantified.

Quantitative Impact of Cell Membrane Fluorescence Labeling on Phagocytosis Measurements in Confrontation Assays.

Phagocytosis is series of steps where the pathogens and the immune cells interact during an invasion. This starts with the adhesion process between the host and pathogen cells, and is followed by the engulfment of the pathogens. Many analytical methods that are applied to characterize phagocytosis based on imaging the host–pathogen confrontation assays rely on the fluorescence labeling of cells. However, the potential effect of the membrane labeling on the quantitative results of the confrontation assays has not been studied in detail. In this study, we determine whether the fluorescence labeling processes themselves influence the results of the phagocytosis measurements. Here, alveolar macrophages, which form one of the most important compartments of the innate immune system, were used as an example of host cells, whereas Aspergillus fumigatus and Lichtheimia corymbifera that cause aspergillosis and mucormycosis, respectively, were studied as examples for pathogens. At first, our study investigated the importance of the sequence of steps of the fixation process when preparing the confrontation assay sample for microscopy studies. Here we showed that applying the fixation agent before the counter-staining causes miscalculations during the determination of the phagocytic measures. Furthermore, we also found that staining the macrophages with various concentrations of DID, as a typical membrane label, in most cases altered the capability of macrophages to phagocytose FITC-stained A. fumigatus and L. corymbifera spores in comparison with unlabeled macrophages. This effect of the DID staining showed a differential character dependent upon the labeling status and the specific type of pathogen. Moreover, labeling the spores of A. fumigatus and L. corymbifera with FITC increased the phagocytic measures during confrontation with unlabeled macrophages when compared to label-free spores. Overall, our study confirms that the staining process itself may significantly manipulate the quantitative outcome of the confrontation assay. As a result of our study, we also developed a user-friendly image analysis tool that analyses confrontation assays both with and without fluorescence labeling of the host cells and of the pathogens. Our image analysis algorithm saves experimental work effort and time, provides more precise results when calculating the phagocytic measures, and delivers a convenient analysis tool for the biologists to monitor host–pathogen interactions as they happen without the artifacts that fluorescence labeling imposes on biological interactions.

Fluorophore-Assisted Click Chemistry through Copper(I) Complexation.

The copper-catalyzed alkyne-azide cycloaddition (CuAAC) is one of the most powerful chemical strategies for selective fluorescent labeling of biomolecules in in vitro or biological systems. In order to accelerate the ligation process and ensure efficient formation of conjugates under diluted conditions, external copper(I) ligands or sophisticated copper(I)-chelating azides are used. This latter strategy, however, increases the bulkiness of the triazole linkage, thus perturbing the biological function or dynamic behavior of the conjugates. In a proof-of-concept study, we investigated the use of an extremely compact fluorophore-based copper(I) chelating azide in order to accelerate the CuAAC with concomitant fluorescence labeling; in our strategy, the fluorophore is able to complex copper(I) species while retaining its photophysical properties. It is believed that this unprecedented approach which was applied for the labeling of a short peptide molecule and the fluorescent labeling of live cells, could be extended to other families of nitrogen-based fluorophores in order to tune both the reaction rate and photophysical characteristics.

Development of a New Nontoxic Thiazole Derivative Dye for Cell Labeling by Using Nanoparticles

Fluorescent labeling of L-929 fibroblast cells was studied by using newly synthesized ligand which was named as 2- N -methylamino-5-(pyrene-1-yl)-thiazole (ABS 40). The maximum fluorescence intensity of ABS 40 was detected at an excitation and emission wavelengths of 350 nm and 400 nm. Fluorescence intensity of the ligand was changed by the addition of DNA from calf thymus (CT-DNA) and human serum albumin (HSA). Both biomolecules and ABS 40 formed complexes that changed the fluorescence intensity of the ligand at the given wavelengths. Cytotoxicity tests were done with XTT analysis. L-929 fibroblast cells were cultured in the presence of ABS 40 to show cell labeling. Results showed that ABS 40 at 318 μM was labeled the cells at an excitation wavelength of 403.7 nm by using confocal microscopy. in vivo cell labeling was also confirmed by using Ct-DNA immobilized iron nanoparticles.

Utilization of Mn‐Doped ZnSe/ZnS Core/Shell Quantum Dots for Rapid Detection of Escherichia coli O157:H7 and Methicillin‐Resistant Staphylococcus aureus

In this study, Mn‐doped ZnSe/ZnS core/shell quantum dots (CSQDs) were synthesized in aqueous solution using polyethylene glycol as a surface stabilizer and successfully applied in the detection of Escherichia coli O157:H7 and methicillin‐resistant Staphylococcus aureus (MRSA) for the first time. The CSQDs were conjugated with anti‐E. coli antibody and anti‐MRSA antibody via protein A supported by 1‐ethyl‐3‐(‐3‐dimethylaminopropyl)carbodiimide hydrochloride for fluorescent labeling of the intact bacterial cells. The detection was performed for the bacterial strains cultivated in Luria‐Bertani liquid medium. The obtained results indicate that E. coli O157:H7 and MRSA can be detected within 30 min at a high sensitivity of 101 CFU/mL. This labeling method based on the highly fluorescent CSQDs may have great potential for use in the food industry to check and prevent outbreaks of foodborne illness.

Efficient Isolation and Long-term Red Fluorescent Nanodia-mond Labeling of Umbilical Cord Mesenchymal Stem Cells for the Effective Differentiation into Hepatocyte-like Cells

Abstract Fluorescent nanodiamond (FND) has been used for long-term cell labeling and in vivo cell tracking because they have good at photostability and biocompatibility. In this study, we evaluate the effect of fluorescent nanodiamond labeling on in vitro culture and differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) into hepatocyte-like cells (HLCs). For hepatic differentiation of hUCMSCs, cells were induced with human hepatocyte growth factor, nicotinamide and Dexamethasone. FND was supplied in two experimental groups with 20 μg/mL and 100 μg/mL in 2 hours. The cell was assessed for FND uptake by laser scan microscopy and flow cytometry methods. The effect of FND on hUCMSCs was evaluated by the cell viability and growth assays as well as the differentiation throughout of morphology alterations or gene expression of anfa-fetoprotein, albumin, and hepatocyte nuclear factor 4α. The results showed that the labeling of hUCMSCs is efficient and easy and there was significant cellular uptake of FND. We did not observe any negative impacts of FND to the cell viability and growth. FND can be utilized for the long-term labeling and tracking of hUCSCs and HLCs in vivo studies.

Cell Mechanics Based Computational Classification of Red Blood Cells Via Machine Intelligence Applied to Morpho-Rheological Markers.

Despite fluorescent cell-labelling being widely employed in biomedical studies, some of its drawbacks are inevitable, with unsuitable fluorescent probes or probes inducing a functional change being the main limitations. Consequently, the demand for and development of label-free methodologies to classify cells is strong and its impact on precision medicine is relevant. Towards this end, high-throughput techniques for cell mechanical phenotyping have been proposed to get a multidimensional biophysical characterization of single cells. With this motivation, our goal here is to investigate the extent to which an unsupervised machine learning methodology, which is applied exclusively on morpho-rheological markers obtained by real-time deformability and fluorescence cytometry (RT-FDC), can address the difficult task of providing label-free discrimination of reticulocytes from mature red blood cells. We focused on this problem, since the characterization of reticulocytes (their percentage and cellular features) in the blood is vital in multiple human disease conditions, especially bone-marrow disorders such as anemia and leukemia. Our approach reports promising label-free results in the classification of reticulocytes from mature red blood cells, and it represents a step forward in the development of high-throughput morpho-rheological-based methodologies for the computational categorization of single cells. Besides, our methodology can be an alternative but also a complementary method to integrate with existing cell-labelling techniques.

Fluorescent Labeling and Quantification of Vesicular ATP Release Using Live Cell Imaging.

Adenosine triphosphate (ATP) is actively transported into vesicles for purinergic neurotransmission by the vesicular nucleotide transporter, VNUT, encoded by the gene, solute carrier 17, member 9 (SLC17A9). In this chapter, methods are described for fluorescent labeling of VNUT positive cells and quantification of vesicular ATP release using live cell imaging. Directions for preparation of viable dissociated neurons and cellular labeling with an antibody against VNUT and for ATP containing synaptic vesicles with fluorescent ATP markers, quinacrine or MANT-ATP, are detailed. Using confocal microscope live cell imaging, cells positive for VNUT can be observed colocalized with fluorescent ATP vesicular markers, which occur as discrete puncta near the cell membrane. Vesicular release, stimulated with a depolarizing, high potassium physiological saline solution induces ATP marker fluorescence reduction at the cell membrane and this can be quantified over time to assess ATP release. Pretreatment with the voltage gated calcium channel blocker, cadmium, blocks depolarization-induced membrane fluorescence changes, suggesting that VNUT-positive neurons release ATP via calcium-dependent exocytosis. This technique may be applied for quantifying vesicular ATP release across the peripheral and central nervous system and is useful for unveiling the intricacies of purinergic neurotransmission.

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Automated sensing and splitting of stem cell colonies on microraft arrays.

Human induced pluripotent stem cells (hiPSCs) are widely used for disease modeling, tissue engineering, and clinical applications. Although the development of new disease-relevant or customized hiPSC lines is of high importance, current automated hiPSC isolation technologies rely largely on the fluorescent labeling of cells, thus limiting the cell line development from many applications. The objective of this research was to develop a platform for high-throughput hiPSC cytometry and splitting that utilized a label-free cell sensing approach. An image analysis pipeline utilizing background subtraction and standard deviation projections was implemented to detect hiPSC colonies from bright-field microscopy data. The pipeline was incorporated into an automated microscopy system coupling quad microraft cell-isolation arrays, computer-based vision, and algorithms for smart decision making and cell sorting. The pipeline exhibited a hiPSC detection specificity of 98% and a sensitivity of 88%, allowing for the successful tracking of growth for hundreds of microcolonies over 7 days. The automated platform split 170 mother colonies from a microarray within 80 min, and the harvested daughter biopsies were expanded into viable hiPSC colonies suitable for downstream assays, such as polymerase chain reaction (PCR) or continued culture. Transmitted light microscopy offers an alternative, label-free modality for isolating hiPSCs, yet its low contrast and specificity for adherent cells remain a challenge for automation. This novel approach to label-free sensing and microcolony subsampling with the preservation of the mother colony holds the potential for hiPSC colony screening based on a wide range of properties including those measurable only by a cell destructive assay.

A standard procedure for lentiviral-mediated labeling of murine mesenchymal stromal cells in vitro.

Tracking of stem cells after transplantation is effectively performed in vivo with imaging systems, assuming the cells are adequately labeled to facilitate their recognition. This study aimed to optimize a protocol for fluorescent labeling of mesenchymal stromal cells (MSCs) in vitro, by using a third-generation lentiviral system. Basically, 293T cells are seeded in high-glucose Dulbecco's modified Eagle medium with 10% FBS one day before transfection. Transfection is done for 24h using a mix of transfer, packaging, regulatory, and envelope plasmids, in molar ratio of 4:2:1:1, respectively. After transfection, the cells are further cultured for two days. During this period, the viral medium is harvested two times, at 24-h intervals, with the first round being stored at 4°C until the second round is completed. The pooled viral medium is frozen in single-use aliquots. MSCs are transduced with 25 multiplicity of infection (MOI) and one day later the cells are passaged at standard seeding density and further grown for three days, when the fluorescence reach the maximum level. Our protocol provides particular experimental details for permanent MSC labeling that makes the procedure highly effective for therapeutic purposes, without affecting the functional properties of stem cells.

Bright Aggregation‐Induced Emission Dots for Dynamic Tracking and Grading of Patient‐Derived Xenografts in Zebrafish

AbstractCancer prognosis will benefit from a scoring system that could grade malignant traits of patient‐derived cells by assessing their growth and metastasis in a living system. Specific tracking of patient‐derived cells requires labeling by contrast agents with good signal‐to‐noise ratio and no specific stain of host tissues. Towards this aim, aggregation‐induced emission (AIE) dots are developed for in vivo cancer tracking with emphasis on reproducible optimized formulation and specific fluorescent labeling of cells that enable enhanced spatial temporal resolution in vivo. The importance of energy‐dependent AIE dots uptake for patient‐derived cell labeling is emphasized to reveal their specific uptake by viable cancer cells. Using optically transparent zebrafish embryo, the ability is demonstrated to follow the engraftment of transplanted AIE dot labeled cells in zebrafish brains over one week. Cells detected outside the brain after 7 d are quantified as metastatic cells. Results from seven clinical samples demonstrate the utility of this methodology to differentiate low engraftment level of benign neoplasms from higher engraftment level and metastasis detected in malignant ovarian cancer specimens. Achieving clinically validated results supports the use of AIE dot labeled patient derived cells in zebrafish xenografts for future cancer prognosis.

Alpha to Beta Cells: A Pathway Towards a Diabetes Cure

Type 1 Diabetes (T1D) is insulin deficiency and hyperglucagonemia due to auto immune destruction of the insulin-secreting beta cells. A viable strategy to treat T1D is to restore the beta cell mass. Here, our approach focuses upon beta cell replacement from alpha cells, which are closely related to beta cells, but not destroyed in T1D. In rodent models containing specific fluorescent genetic lineage labeling of alpha and beta cells, we identified a group of cells at the islet periphery that appear to be beta cells that are transdifferentiated from alpha cells (AB). Preliminary live cell imaging and electrophysiology studies suggest that these AB cells acquire a typical beta cell glucose response, which suggests that beta cell regeneration from endogenous alpha cell populations may be a viable TID cure approach. In human islets, we also discovered endogenous cells expressing both insulin and glucagon. The dynamics of these bi-hormonal cells in terms of glucose response and hormone (insulin and/or glucagon) secretion are yet to be elucidated. We are performing imaging based islet studies that utilize virally delivered far red fluorescent secretory pathway fluorescent labels to determine the glucose response and inulin/ glucagon secretion dynamics of these bi hormonal cells. Our single cell islet secretion assays will also be used in combination with alpha and beta cell specific RCaMP1h and GCaMP6 Ca2+ response indicators in order to also understand how Ca2+ couples with secretion in these cells types. Findings from this study will advance our understanding in these unique rodent and human cell types. The long term goal study goal is to target these unique cell populations for conversion into functional beta cells.