Cell-permeable Dye Research Articles

Cysteine Leukotriene Receptor Antagonist-Montelukast Effects on Diabetic Retinal Microvascular Endothelial Cells Curtail Autophagy.

Diabetic macular edema (DME) is the primary cause of vision impairment in diabetic retinopathy (DR) patients. A previous study has shown the efficacy of montelukast, a cysteinyl leukotriene receptor (CysLTR)1 antagonist, in a diabetic mouse model. This study aims to understand the CysLTR1 signaling in retinal endothelial cells and the impact of montelukast. Primary human retinal microvascular endothelial cells (HRECs) challenged with 20 ng/mL TNF-α and 30mM D-glucose (D-glu) for six to 24 hours served as a model of endothelial activation. HRECs were incubated with L-glucose (L-glu) as an osmotic control. CysLTR1 knockdown and montelukast pretreatment assessed CysLTR1 antagonism. Gene expression, protein expression, and cell-permeable dyes were utilized to measure autophagy and inflammation. Transendothelial electrical resistance (TER) and transendothelial migration of mononuclear leukocytes across HRECs monolayer were measured as a functional assessment of vascular permeability. Endothelial activation induced by hyperglycemia and inflammation increased CysLTR1 expression, triggering autophagy within two to six hours, IL-1β production, loss of junction integrity, decreased TER, and increased leukocyte migration within six to 24 hours. Pretreatment with montelukast effectively alleviated these effects, demonstrating its dependence on CysLTR1. Dysfunctional retinal endothelium initiates a self-reinforcing loop of inflammation, autophagy, and compromised integrity associated with heightened CysLTR1 levels. The antagonistic effect of montelukast against CysLTR1 effectively mitigates these detrimental changes. This study reveals CysLTR1 as a potential therapeutic target in treating DME and offers a novel strategy to mitigate detrimental changes in DR.

Click Chemistry with Cell-Permeable Fluorophores Expands the Choice of Bioorthogonal Markers for Two-Color Live-Cell STED Nanoscopy.

STED nanoscopy allows for the direct observation of dynamic processes in living cells and tissues with diffraction-unlimited resolution. Although fluorescent proteins can be used for STED imaging, these labels are often outperformed in photostability by organic fluorescent dyes. This feature is especially crucial for time-lapse imaging. Unlike fluorescent proteins, organic fluorophores cannot be genetically fused to a target protein but require different labeling strategies. To achieve simultaneous imaging of more than one protein in the interior of the cell with organic fluorophores, bioorthogonal labeling techniques and cell-permeable dyes are needed. In addition, the fluorophores should preferentially emit in the red spectral range to reduce the potential phototoxic effects that can be induced by the STED light, which further restricts the choice of suitable markers. In this work, we selected five different cell-permeable organic dyes that fulfill all of the above requirements and applied them for SPIEDAC click labeling inside living cells. By combining click-chemistry-based protein labeling with other orthogonal and highly specific labeling methods, we demonstrate two-color STED imaging of different target structures in living specimens using different dye pairs. The excellent photostability of the dyes enables STED imaging for up to 60 frames, allowing the observation of dynamic processes in living cells over extended time periods at super-resolution.

Repli-seq Sample Preparation using Cell Sorting with Cell-Permeant Dyes.

Replication timing is significantly correlated with gene expression and chromatin organization, changes dynamically during cell differentiation, and is altered in diseased states. Genome-wide analysis of replication timing is performed in actively replicating cells by Repli-seq. Current methods for Repli-seq require cells to be fixed in large numbers. This is a barrier for sample types that are sensitive to fixation or are in very limited numbers. In this article, we outline optimized methods to process live cells and intact nuclei for Repli-seq. Our protocol enables the processing of a smaller number of cells per sample and reduces processing time and sample loss while obtaining high-quality data. Further, for samples that tend to form clumps and are difficult to dissociate into a single-cell suspension, we also outline methods for isolation, staining, and processing of nuclei for Repli-seq. The Repli-seq data obtained from live cells and intact nuclei are comparable to those obtained from the standard protocols. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Live cell isolation and staining Alternate Protocol: Nuclei isolation and staining.

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic β-Cells.

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional β-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in β-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify β-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for β-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in β-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous β-cell models.

A Triphosphate Pronucleotide (TriPPPro)Reporter with Optimized Cell-Permeable Dyes for Metabolic Labeling of Cellular and Viral DNA in Living Cells.

The metabolic labeling of nucleic acids in living cells is highly desirable to track the dynamics of nucleic acid metabolism in real-time and has the potential to provide novel insights into cellular biology as well as pathogen-host interactions. Catalyst-free inverse electron demand Diels-Alder reactions (iEDDA) with nucleosides carrying highly reactive moieties such as axial 2-trans-cyclooctene (2TCOa) would be an ideal tool to allow intracellular labeling of DNA. However, cellular kinase phosphorylation of the modified nucleosides is needed after cellular uptake as triphosphates are not membrane permeable. Unfortunately, the narrow substrate window of most endogenous kinases limits the use of highly reactive moieties. Here, we apply our TriPPPro (triphosphate pronucleotide) approach to directly deliver a highly reactive 2TCOa-modified 2'-deoxycytidine triphosphate reporter into living cells. We show that this nucleoside triphosphate is metabolically incorporated into de novo synthesized cellular and viral DNA and can be labeled with highly reactive and cell-permeable fluorescent dye-tetrazine conjugates via iEDDA to visualize DNA in living cells directly. Thus, we present the first comprehensive method for live-cell imaging of cellular and viral nucleic acids using a two-step labeling approach.

Fluorogenic and Cell-Permeable Rhodamine Dyes for High-Contrast Live-Cell Protein Labeling in Bioimaging and Biosensing.

The advancement of fluorescence microscopy techniques has opened up new opportunities for visualizing proteins and unraveling their functions in living biological systems. Small-molecule organic dyes, which possess exceptional photophysical properties, small size, and high photostability, serve as powerful fluorescent reporters in protein imaging. However, achieving high-contrast live-cell labeling of target proteins with conventional organic dyes remains a considerable challenge in bioimaging and biosensing due to their inadequate cell permeability and high background signal. Over the past decade, a novel generation of fluorogenic and cell-permeable dyes has been developed, which have substantially improved live-cell protein labeling by fine-tuning the reversible equilibrium between a cell-permeable, nonfluorescent spirocyclic state (unbound) and a fluorescent zwitterion (protein-bound) of rhodamines. In this review, we present the mechanism and design strategies of these fluorogenic and cell-permeable rhodamines, as well as their applications in bioimaging and biosensing.

Sodium butyrate supresses malignant human mast cell proliferation, downregulates expression of KIT and promotes differentiation.

Sodium butyrate (NaBu) is a class I histone deacetylase inhibitor (HDACi) that can impede the proliferation of transformed cells. Although some HDACi downregulate the expression of the stem cell factor receptor (KIT/CD117), the effect of NaBu on KIT expression and human mast cell proliferation requires further elucidation. In this study, we examined the effects of NaBu on three transformed human mast cell lines, HMC-1.1, HMC-1.2 and LAD2. NaBu (100 µM) inhibited the proliferation and metabolic activity of all three cell lines without significantly affecting their viability, suggesting that although the cells had ceased to divide, they were not yet undergoing apoptosis. Cell cycle analysis using the cell-permeant dye, propidium iodide, indicated that NaBu significantly blocked the cell cycle progression of HMC-1.1 and HMC-1.2 from G1 to G2/M phases. Furthermore, NaBu downregulated the expression of C-KIT mRNA and KIT protein expression in all three cell lines, but this effect was most significant in the HMC-1.1 and HMC-1.2, both of which harbour activating mutations in KIT, which proliferate more rapidly than LAD2. These data support earlier observations showing that human mast cell lines are sensitive to histone deacetylase inhibition. However, our data presents the novel observation that inhibition of cell proliferation by NaBu was not associated with a loss in cell viability but rather an arrest of the cell cycle. Higher concentrations of NaBu led to modest increases in histamine content, tryptase expression, and granularity. In conclusion, NaBu treatment of human mast cell lines led to a modest enhancement of the hallmarks of mature mast cells.

In-droplet cell lysis of AC16 human cardiomyocyte cells via surface acoustic waves.

Although several lysis methods are available, biomedical applications are pushing the demand for miniaturised systems and thus for new ways to lyse cells in small volumes. In this work, we demonstrate in-droplet cell lysis of AC16 human cardiomyocyte cells in 20 μL droplets using high frequency surface acoustic waves. The acoustic streaming leads to high shear flow creating porous or breaking the cell membrane and releasing intracellular material. Contrary to previous work where the lysis efficiency is measured by a cell-permeant dye that can be used to determine cell viability, here we propose to quantify the DNA extracted from the cells as a measure of the lysis efficiency. This reagent-free method provides a valuable cell lysis alternative for many biological and biomedical applications, particularly for the development of point-of-care platforms.

Enhancing α-CD19 CAR T Antitumor Efficacy Using the BH3 Mimetic, Venetoclax

Introduction Clinical outcomes of anti-CD19 chimeric antigen receptor T cell (CAR T) therapy for the treatment of relapsed/refractory Diffuse Large B Cell lymphoma (r/r DLBCL) must be improved, as patients undergoing CAR T therapy can expect a cure rate of 41% and median survival of just 6.3 months if unresponsive. Poor clinical responses to CAR T therapy and a small window of survival highlights the critical need for improvement of CAR T therapy in r/r DLBCL. Our lab previously revealed synergy between the Bcl-2 inhibitor, Venetoclax, and α-CD19 CAR Ts. Though the role of Bcl-2 inhibition within B cell lymphomas is well characterized, further investigation is needed to understand its impact within effector cell populations. Our lab previously established a role for Bcl-2 inhibition in promoting the differentiation of bulk CD8+ CAR T into CD8+ CAR TStem Cell Memory (TSCM) subsets, decreasing the frequency of CD4+ CAR TRegulatory subsets, and increasing cytotoxicity of CAR T when co-cultured with rituximab/chemoresistant malignant B cells, Raji4RH. Here, our lab further elucidates the molecular mechanisms driving these improvements in CAR T quality as a consequence of Bcl-2 inhibition. MethodsTransduction was performed on healthy donor T cells with lentiviral vectors encoding for 2nd generation α-CD19 CAR constructs containing CD28 (28ζ) or 4-1BB (BBζ) costimulatory domain, 24 hours after activation with αCD3/CD28 TransACT (Miltenyi). Cytotoxicity assays were performed by labeling target cells with a lipophilic dye (Sigma-Aldrich), then incubated with GFP+ CAR T at various effector:target ratios and Venetoclax concentrations for 24 hours in 96 well U-bottom plates. Sample wells were stained with viability dye and acquired on BD LSR II cytometer. Mitochondrial membrane potential (ΨM) was assessed using a cell-permeant fluorescent probe, TMRM (Thermofisher), which was added to immunophenotype panels. CAR T immunophenotype was identified using markers for viability, CD3, CD4, CD8, CD45RA, CD45RO, CD62L, CCR7, CD25, CD95, and TMRM. Frequencies of T cell memory subsets were calculated alongside changes in ΨM. B-cell marker expression was assessed for several ABC and GCB DLBCL lines. Antibodies against CD10, CD20, CD22, CD138, and CD19 were used to establish B-lineage epitope density within our target populations. Proliferation tracking was performed using cell-permeant dye, CellTrace Blue (Thermofisher). Bulk untransduced, CD28, or 4-1BB CAR T were stained with tracking dye, activated for 24 hours by co-culture with CD19+ cells, then isolated using CD19+ Microbead positive selection (Miltenyi Biotec). Then, the untransduced control and CAR T fractions were cultured in Venetoclax for up to 96 hours. Samples were then collected and stained with naïve and memory T cell markers and a viability dye. Results Untransduced control, 28ζ, and BBζ CAR T were exposed to Venetoclax during co-culture with DLBCL lines expressing varying densities of CD19 epitope. A significant concentration-dependent decrease in ΨM was observed for each CAR construct. CAR viability did not decrease more than 5% among each concentration and cell line tested. As Venetoclax concentration increases, larger frequencies of CD8+ TNaive and TSCM are observed. Therefore, we conclude an inverse relationship between ΨM and differentiation into CD8+ TSCM cells. CD19+ epitope density does not significantly impact CAR memory phenotype, ΨM, or cytolytic capacity. To uncover the progenitor cell type populating Venetoclax-exposed CD8+ CAR TSCM cells, we tracked dye loss between subsets TNaive, TSCM, and TTotal. After 96 hours of culture, it was apparent that the TSCM cohort was not significantly proliferating, however the TNaive subset experienced the most dye loss, and accounted for observed dye loss within the TTotal cohort. We conclude that Venetoclax encourages differentiation from TNaive cell to TSCM cell. Conclusion We reveal intrinsic changes within the CAR T as a result of Bcl-2 inhibition; and propose that these changes are due to manipulation of mitochondria, allowing for differentiation into a more quiescent, stem-like cell type. Low ΨM has been linked to memory T cell phenotype and increased anti-tumor function in vivo. We seek to uncover the metabolic mechanisms which regulate the observed changes within CAR Ts as a result of Bcl-2 inhibition. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Photoinduced Carbene for Effective Photodynamic Therapy Against Hypoxic Cancer Cells

Photoinduced Carbene for Effective Photodynamic Therapy Against Hypoxic Cancer Cells

Monitoring Changes in Intracellular Reactive Oxygen Species Levels in Response to Histone Deacetylase Inhibitors.

Reactive oxygen species (ROS) are induced by several chemotherapeutics. In this protocol, we describe a flow cytometry-based method for the analysis of the intracellular levels of ROS in vital leukemic cells in response to the histone deacetylase inhibitor vorinostat. This measurement of ROS using the cell-permeable dye CM-H2DCFDA indicates intracellular oxidative stress.

Histochemical targeting: Combining plasma immersion ion implantation and histochemical probes to target magnetic particles to intracellular cytological features

Paramagnetic microparticles are effective tools for cell purification, diagnostics, imaging and drug delivery and have been used for over 30 years to separate cell types from complex mixtures. The targeting of magnetic particles to cells and tissues is dependent upon the use of antibodies and adhesion molecules as targeting modalities. Here, we present a novel paradigm for the targeting of microparticles to cells according to intracellular cytological characteristics. Cell-permeable histochemical dyes, when covalently conjugated to magnetic microparticles, following plasma immersion ion implantation (PIII) imbue the particles with cell targeting properties in a process we term histochemical targeting. PIII treatment and conjugation to the lipophilic dyes Nile Red or Nile Blue led to changes in microparticle size, surface charge and lipophilicity. PIII-treated microparticles covalently conjugated with Nile Red, crossed the live cell membrane and homed to intracellular neutral lipid droplets. Nile Blue-conjugated particles crossed live cell membranes and remained in the cytosol where free fatty acids localize. Magnetic cell sorting could be performed on cultured adipocytes, as well as rat liver and adrenal cell suspensions using particles conjugated to Nile Red or Nile Blue. Taken together, our findings demonstrate that histochemical probes can be covalently conjugated to magnetic particles following PIII treatment resulting in intracellular targeting. With a vast armamentarium of cell-permeable probes available, histochemical targeting could be applied to many fields requiring magnetic particle delivery to specific sites or isolation of particular cell types.

Ultrastructure Expansion Microscopy (U-ExM) in Trypanosoma cruzi: localization of tubulin isoforms and isotypes.

Ultrastructure Expansion Microscopy (U-ExM) is a recently developed technique that enables the increase of the spatial resolution within a cell or a tissue for microscopic imaging by physically expanding the sample. For the first time, I report a detailed protocol validating the use of U-ExM in Trypanosoma cruzi and quantifying the expansion factors of different subcellular compartments. I was able to determine the localization patterns of different tubulin isoforms, such as α-tubulin and β-tubulin. Also, I immunolocalized acetylated and tyrosinated α-tubulin isotypes in epimastigotes and use mitochondrial cell-permeable dyes to identify this organelle. Finally, U-ExM was also performed in trypomastigotes and amastigotes validating this technique in all life cycle stages of T. cruzi.

Supercharged MPNs? Automated Determination of High-Throughput Most Probable Number (htMPN) Using Chip-Based 3D Digital PCR.

Surface plating on agar and most probable number (MPN) are the standard methods for determining bacterial viability but both have limitations. Here we present a novel cell count method, high-throughput MPN (htMPN), that uses a chip-based digital PCR instrument to accelerate and to improve the quantification of viable or sublethally injured cells. This method tracks growth of up to 20,000 individual bacterial cells on a single chip. Single cells were grown in the individual wells of the chip at their optimal temperature until the cell density was high enough to detect the fluorescent signal with cell-permeant or cell-impermeant DNA-intercalating fluorescent dyes. This method based on microfluidic devices implemented in digital PCR equipment was equivalent to surface plating in determining cell counts of Escherichia coli, Salmonella enterica serovar Typhimurium, Fructilactobacillus sanfranciscensis, Pseudomonas putida, and vegetative cells but not spores of Bacillus subtilis. Viable E. coli could be enumerated within 7 h. Culture of strict aerobes was restricted to strains that are capable of nitrate respiration; organisms requiring complex media that also contain double-stranded DNA were detected after treatment of growth media with DNase before inoculation. Our approach not only monitors the frequency distribution of bacterial growth and determines cell counts with high reliability but also detected heat-injured cells of S. Typhimurium that escaped detection by the surface plating. Overall, the method accelerates detection of viable bacterial cells, facilitates automation, and offers new possibilities for the analysis of individual bacterial cells. IMPORTANCE htMPN uses chip-based fluorescence acquisition and is a simple and compact tool for automatic viable cell enumeration with applications in microbiological research. This method applies to a wide range of anaerobic or facultative anaerobic species and improves accuracy by reducing the number of pipetting steps. In addition, the method offers an additional tool for single-cell microbiology. The single cell time-to-detection times have been used as an important criterion for the physiological state of bacterial cells after sublethal stress, and htMPNs support the acquisition of such data with an unprecedented number of cells. In particular, htMPN provides an anaerobic environment and enables a long incubation time to increase the recovery rate of sublethally injured cells. Given its reproducibility and reliability, our approach can potentially be applied to quantify viable cells in samples from environmental, clinical, or food samples to reduce the risk of underestimation of the number of viable bacterial cells.

An Ex Vivo Model of Oxidatively Stressed Red Blood Cells Demonstrates a Role for Exogenous Amino Acids in Enhancing Red Blood Cell Function and Morphology

The capacity of mature red blood cells (RBCs) to respond to oxidative stress is limited due to lack of a full complement of organelles and as such, when faced with an oxidative environment, they rely on their endogenous antioxidant capacity (including superoxide dismutase, catalase, peroxiredoxin and glutathione) to protect against cellular damage. Low blood glutathione activity has been reported in several red cell disorders leading to increased oxidative stress. Targeting oxidative stress has thus been proposed as a secondary treatment in multiple anemia-causing diseases, such as sickle cell disease (SCD) and malaria, although its overall efficacy remains unclear. As glutathione itself is not permeable through the RBC membrane, treatment with cell-permeable amino acid precursors of glutathione (glutamine, cysteine and/or glycine) is a potential strategy to expand the RBC's antioxidant capacity and alleviate oxidative stress. Indeed, L-glutamine has recently been approved as a therapeutic for SCD, although the mechanistic basis for its effect is not clear. To fill this gap in our understanding, we performed detailed characterization of biophysical phenotype, morphology, and intracellular redox environment of oxidatively stressed RBCs in environments with varying amounts of available precursor amino acids.To assess the impact of exogenous amino acid precursors on the RBC's glutathione antioxidant capacity, we exposed mature RBCs from healthy adults to hydrogen peroxide (H 2O 2) and co-incubated with media that included glutamine, cysteine, and/or glycine. As catalase has the ability to scavenge high levels of exogenously fluxed H 2O 2, we performed these experiments using sodium azide to block catalase activity, enabling us to model oxidatively stressed RBCs. We performed osmotic gradient ektacytometry to quantify RBC deformability and hydration status, and assessed RBC morphology using osmotic-adjusted fixation techniques and scanning electron microscopy. As previously documented, H 2O 2 exposure in sodium azide-treated healthy RBCs was associated with decreased deformability, decreased hydration and increased numbers of echinocytes in a dose-dependent manner. We monitored red cell phenotypic changes following co-incubation with glutamine, cysteine, and/or glycine individually and in combination to test whether these amino acids extended the RBC's antioxidant abilities and contributed to improved function and morphology. We found that supplementation with all three amino acids in combination significantly improved both deformability and hydration of H 2O 2-stressed RBCs, as opposed to treatment with glutamine alone. To directly assess whether the exogenous amino acids were in fact contributing to less intracellular oxidative stress in RBCs, we quantified intracellular reactive oxygen species (ROS) using 2‘, 7‘ -dichlorofluorescein diacetate (DCFDA), a cell permeable dye used to measure ROS production. As expected, H 2O 2 exposure alone was associated with elevated intracellular ROS inside RBCs in both a time- and dose-dependent manner. Supplementation with the three amino acid cocktail during H 2O 2 stress resulted in a reduction in the level of intracellular ROS activity.In summary, we documented that exogenous added amino acids reduce oxidative damage in RBCs and we hypothesize that this protection occurs via the glutathione antioxidant pathways. In future studies, we plan to investigate the impact of exogenous amino acids on sickled and irreversibly sickled RBCs (ISCs) in the context of SCD, and on uninfected and infected RBCs in the context of malaria. DisclosuresKim-Shapiro: Beverage Operations LLC: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: co-inventor on a patent related to the use of nitrite under cardiovascular conditions, and a co-author on patents related to treatment of hemolysis.

Modulation of Intracellular ROS and Senescence-Associated Phenotypes of Xenopus Oocytes and Eggs by Selective Antioxidants.

Aging of oocytes and eggs diminishes their reproductive and developmental potential. It has been demonstrated previously that reactive oxygen species (ROS) contribute to accelerated aging of various cells. In the present study, we measured intracellular levels of ROS and investigated effects of several selective antioxidants (AOXs) on the viability and functional activity of aging oocytes and eggs of the African clawed frog Xenopus laevis. The fluorescent cell-permeable dye DCFDA, which is widely employed for ROS detection in cultured mammalian cells, was used to monitor ROS levels in the fresh and bench-aged oocytes and eggs by an optimized protocol. It was found that intracellular ROS contents were increased in frog oocytes and eggs aged for 48 h. It was further demonstrated using selective cell-permeable AOXs targeting different ROS-generating mechanisms, that the major source of ROS in Xenopus oocytes and eggs is the plasma membrane NADPH oxidase, and that mitochondrial generation contributes to the intracellular ROS content to a lesser extent. Targeted inhibition of NADPH oxidase with a natural organic compound apocynin reduced ROS levels significantly in Xenopus oocytes and eggs, maintained their normal phenotype and supported their functional competence. To our knowledge this is the first report concerning beneficial effects of apocynin on the isolated gamete cells, such as oocytes and eggs.

Myofibrillar Structural Variability Underlies Contractile Function in Stem Cell-Derived Cardiomyocytes

SummaryDisease modeling and pharmaceutical testing using cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) requires accurate assessment of contractile function. Micropatterning iPSC-CMs on elastic substrates controls cell shape and alignment to enable contractile studies, but determinants of intrinsic variability in this system have been incompletely characterized. The objective of this study was to determine the impact of myofibrillar structure on contractile function in iPSC-CMs. Automated analysis of micropatterned iPSC-CMs labeled with a cell-permeant F-actin dye revealed that myofibrillar abundance is widely variable among iPSC-CMs and strongly correlates with contractile function. This variability is not reduced by subcloning from single iPSCs and is independent of the iPSC-CM purification method. Controlling for myofibrillar structure reduces false-positive findings related to batch effect and improves sensitivity for pharmacologic testing and disease modeling. This analysis provides compelling evidence that myofibrillar structure should be assessed concurrently in studies investigating contractile function in iPSC-CMs.

Ex-vivo Microtubule Stability Assay Using Drosophila Wing Disc.

Regulation of microtubule stability is crucial for diverse biological processes, including cell division, morphogenesis, and signaling. Various in vitro assays for microtubule stability have been developed to identify and characterize proteins involved in controlling microtubule stability. Here, we introduce a simple ex-vivo assay for identifying potential microtubule regulators in the wing imaginal disc of Drosophila melanogaster. This assay utilizes silicon rhodamine-tubulin (SiR-Tub) as a cell-permeable fluorogenic dye for labeling microtubules. In an attempt to increase the sensitivity of the screen, we designed an assay using a sensitized microtubule condition. Wing discs are treated with SiR-Tub followed by demecolcine, a microtubule inhibitor, to partially label impaired microtubules. Under this sensitized condition, we can test whether overexpression or downregulation of a gene can enhance or suppress the weakened SiR-Tub labeling. This assay allows highly sensitive detection of microtubules in developing larval tissues. Hence, it provides a useful tool for identifying new microtubule regulators in both unfixed and fixed imaginal discs in Drosophila. This strategy may also be applied to characterize microtubule regulators in tissues from other model organisms. Graphic abstract: Graphical summary of Ex-vivo microtubule stability assay using Drosophila wing disc.

Universal Single-Residue Terminal Labels for Fluorescent Live Cell Imaging of Microproteins.

Genetically encoded fluorescent tags for visualization of proteins in living cells add six to several hundred amino acids to the protein of interest. While suitable for most proteins, common tags easily match and exceed the size of microproteins of 60 amino acids or less. The added molecular weight and structure of such fluorescent tag may thus significantly affect in vivo biophysical and biochemical properties of microproteins. Here, we develop single-residue terminal labeling (STELLA) tags that introduce a single noncanonical amino acid either at the N- or C-terminus of a protein or microprotein of interest for subsequent specific fluorescent labeling. Efficient terminal noncanonical amino acid mutagenesis is achieved using a precursor tag that is tracelessly cleaved. Subsequent selective bioorthogonal reaction with a cell-permeable organic dye enables live cell imaging of microproteins with minimal perturbation of their native sequence. The use of terminal residues for labeling provides a universally applicable and easily scalable strategy, which avoids alteration of the core sequence of the microprotein.

Rat bone healing induced by natural nanocrystalline carbonated hydroxyapatite in combination with human amniotic fluid stem cells (AFSCs).

The present study was aimed at identifying a new scaffold/stem cell combination useful to treat large bone defects. Human amniotic fluid stem cells (AFSCs) were expanded in vitro, labeled with a fluorescent cell-permeable dye (PKH26) and transplanted in vivo in a femoral injured rat model. The femoral defect was left untreated (control rats) or filled with hydroxyapatite (HA; natural nanocrystalline carbonated hydroxyapatite-Orthoss®) scaffold alone or loaded with PKH26-labeled AFSCs. All animals were killed 3 weeks after implantation. Both gross anatomy and histological observations revealed a major bone regenerative response in rat specimens treated with HA scaffold, alone or supplemented with AFSCs. Samples injected with HA plus AFSCs displayed the presence of abundant fibrotic tissue, the formation of periosteal woven bone, and an increased presence of blood vessels in the bone marrow, with still fluorescent AFSCs in close proximity. These observations provide evidence that natural HA plus AFSCs represents a promising alternative therapeutic strategy to autologous bone grafting procedures.