Cellular Fluorescence Research Articles

Transcription Factor RUNX1 Regulates Factor FXIIIA Subunit (F13A1) Expression in Megakaryocytic Cells and Platelet F13A1 Expression is Downregulated in RUNX1 Haplodeficiency

Plasma Factor XIII, a transglutaminase, is a complex of two A (gene F13A1) chains with catalytic activity and two B chains (gene F13B). FXIII induces clot stabilization and retraction via cross-linking of fibrin monomers and other proteins. Factor XIII deficiency in plasma causes a major bleeding disorder with impaired clot retraction. FXIIIA is synthesized in megakaryocytes, and is abundant in platelets. It is expressed on activated platelets and cross-links proteins on platelet surface. Little is known regarding the regulation of F13A1 in megakaryocytes and platelets. RUNX1 is a major hematopoietic transcription factor and regulates expression of numerous genes in megakaryocytes and platelets. Patients with RUNX1 haplodeficiency (RHD) with heterozygous mutations have thrombocytopenia, platelet dysfunction with impaired responses to activation, and predisposition to myeloid malignancies. Platelet expression profiling using Affymetrix microarrays of a patient (P1) with a heterozygous RUNX1 mutation (c.969-323G>T) revealed decreased F13A expression, which encodes for subunit A of FXIII: fold-change 0.30; p=0.006; patient profiled twice 10 months apart and compared to 6 healthy subjects. These findings were confirmed by qPCR With RT-PCR platelet F13A mRNA expression was decreased to ~ 20% of mean expression in 4 subjects. Platelet F13A protein expression was decreased by ~50% as assessed by corrected total cellular fluorescence imaging. Platelet F13A mRNA expression was also decreased (to less than 20% of expression in 5 controls) in two siblings (P2 and P3 ages 8 and 3 years) from an unrelated family with a heterozygous RUNX1 mutation 508+1G>A). Plasma FXIII was normal in the patient P1. We studied the regulation of F13A by RUNX1 in human erythroleukemia (HEL) cells treated with phorbol myristate acetate (PMA) to induce megakaryocytic transformation. In silico analyses of the 5' upstream region of F13A showed 7 RUNX1 consensus sites within 545bp from ATG. Chromatin immunoprecipitation studies performed using HEL cells and RUNX1 antibody showed that that RUNX1 binds in the region encompassing site 4 and sites 5-7. In parallel, electrophoretic mobility shift assay (EMSA) studies showed binding of nuclear protein to labeled DNA probes with sites 1, 4, 5, 6, and 7. RUNX1 antibody produced a "supershift" with addition suggesting RUNX1 binding. Site-directed mutagenesis of the RUNX1 binding sites in F13A1 promoter decreased promoter activity in luciferase reporter studies in HEL cells, providing evidence that the binding sites are functional. Further, overexpression of RUNX1 in HEL cells increased F13A1 promoter activity and protein while siRNA RUNX1 knockdown reduced F13A1 protein. We assessed clot retraction over 90 min at 37oC in the patient P1 and his daughter (also with RUNX1 mutation) in citrated whole blood following addition of tissue factor (1 pM) and CaCl2 (1 mM), and in washed platelets resuspended in platelet poor plasma from a healthy subject supplemented with added fibrinogen (0.5 mg/mL) and stimulated with 0.5 U/mL thrombin in the presence of CaCl2. These preliminary studies suggested that clot retraction was slower in the patient P1 compared to the controls, particularly in studies with platelet suspensions. Conclusions: Overall, these studies provide the first evidence that hematopoietic transcription factor RUNX1 regulates expression of F13A1 in megakaryocytic cells and that platelet expression of F13A1 is decreased in platelets from patients with RUNX1 haplodeficiency. The decreased expression of F13A1 in RHD may contribute to the platelet dysfunction in RHD. These findings are particularly interesting because they reflect regulation of a coagulation protein (FXIIIA) by hematopoietic transcription factor RUNX1. Disclosures Lambert: Dova: Consultancy, Membership on an entity's Board of Directors or advisory committees; Principia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Shionogi: Consultancy; Educational Concepts in Medicine: Consultancy; Octapharma: Consultancy, Research Funding; Bayer: Consultancy; Platelet Disorder Support Association (PDSA): Consultancy; 22qSociety: Consultancy; ITP Australia: Consultancy; CdLS Foundation: Consultancy; RDMD ITP study: Consultancy; Sysmex: Research Funding; AstraZeneca: Research Funding; Argenix: Consultancy; ClinGen: Honoraria.

Apelin-13 regulates angiotensin ii-induced Cx43 downregulation and autophagy via the AMPK/mTOR signaling pathway in HL-1 cells.

Atrial fibrillation is associated with atrial remodeling, in which connexin 43 (Cx43) and cell hypertrophy play important roles. In this study, apelin-13, an aliphatic peptide, was used to explore the protective effects of the adenosine monophosphate-activated protein kinase (AMPK)/mTOR signaling pathway on Cx43 expression and autophagy, using murine atrial HL-1 cells. The expression of Cx43, AMPK, B-type natriuretic peptide (BNP) and pathway-related proteins was detected by Western blot analysis. Cellular fluorescence imaging was used to visualize Cx43 distribution and the cytoskeleton. Our results showed that the Cx43 expression was significantly decreased in HL-1 cells treated with angiotensin II but increased in cells additionally treated with apelin-13. Meanwhile, apelin-13 decreased BNP expression and increased AMPK expression. However, the expression of Cx43 and LC3 increased by apelin-13 was inhibited by treatment with compound C, an AMPK inhibitor. In addition, rapamycin, an mTOR inhibitor, promoted the development of autophagy, further inhibited the protective effect on Cx43 expression and increased cell hypertrophy. Thus, apelin-13 enhances Cx43 expression and autophagy via the AMPK/mTOR signaling pathway, and serving as a potential therapeutic target for atrial fibrillation.

Flow Cytometric Measurement of Efflux in Candida Species.

A technique to assess the ability of distinct Candida strains to efflux substrates, as well as to compare the effectiveness of efflux inhibitors, is important for analysis of antifungal drug resistance mechanisms and the mode of action of antifungals. We describe a method that measures the ability of Candida species to extrude the fluorescent dye Nile red as an output for efflux activity. This involves exposing cells to Nile red and using flow cytometry to quantify cellular fluorescence, enabling numerous samples to be processed in a limited time frame. This protocol provides a simple, yet effective method for quantifying efflux in drug-resistant Candida species. © 2020 Wiley Periodicals LLC Basic Protocol 1: Growth and sample preparation of stained Candida Basic Protocol 2: Quantitative measurement of fluorescence by flow cytometry Alternate Protocol: Qualitative determination of fluorescence using microscopy.

5-Aminolevulinic acid for fluorescence-guided surgery in pancreatic cancer: Cellular transport and fluorescence quantification studies

5-Aminolevulinic acid (ALA) is a potential contrast agent for fluorescence-guided surgery in pancreatic ductal adenocarcinoma (PDAC). However, factors influencing ALA uptake in PDAC have not been adequately assessed. We investigated ALA-induced porphyrin fluorescence in PDAC cell lines CFPAC-1 and PANC-1 and pancreatic ductal cell line H6c7 following incubation with 0.25–1.0 mM ALA for 4–48 h. Fluorescence was assessed qualitatively by microscopy and quantitatively by plate reader and flow cytometry. Haem biosynthesis enzymes and transporters were measured by quantitative polymerase chain reaction (qPCR). CFPAC-1 cells exhibited intense fluorescence under microscopy at low concentrations whereas PANC-1 cells and pancreatic ductal cell line H6c7 showed much lower fluorescence. Quantitative fluorescence studies demonstrated fluorescence saturation in the two PDAC cell lines at 0.5 mM ALA, whereas H6c7 cells showed increasing fluorescence with increasing ALA. Based on the PDAC:H6c7 fluorescence ratio studies, lower ALA concentrations provide better contrast between PDAC and benign pancreatic cells. Studies with qPCR showed upregulation of ALA influx transporter PEPT1 in CFPAC-1, whereas PANC-1 upregulated the efflux transporter ABCG2. We conclude that PEPT1 and ABCG2 expression may be key contributory factors for variability in ALA-induced fluorescence in PDAC.

Gold embedded chitosan nanoparticles with cell membrane mimetic polymer coating for pH-sensitive controlled drug release and cellular fluorescence imaging.

In this work, gold embedded chitosan nanoparticles (Au@CS NPs) were fabricated by a one-pot method. The benzaldehyde-terminated poly[(2-methacryloyloxy) ethyl phosphorylcholine] (PMPC) was applied to modification of the gold doped chitosan nanoparticles. The obtained Au@CS-PMPC NPs had the diameter of 135 nm with a narrow distribution. The size of the Au@CS-PMPC NPs, as well as the size of the embedded gold NPs, might be well-controlled by adjusting the feeding ratio between chitosan and HAuCl4. Furthermore, the Au@CS-PMPC NPs showed increased colloidal stability, high drug loading content, pH-responsive drug release, excellent biocompatibility and bright fluorescence emission. The results demonstrated that Au@CS-PMPC NPs showed a great potential for tumor therapy via the combination advantages of pH-sensitive controlled drug release and cellular fluorescence imaging.

Human Nontumorigenic Microglia Synthesize Strongly Fluorescent Au/Fe Nanoclusters, Retaining Bioavailability.

The ability for cells to self-synthesize metal-core nanoclusters (mcNCs) offers increased imaging and identification opportunities. To date, much work has been done illustrating the ability for human tumorigenic cell lines to synthesize mcNCs; however, this has not been illustrated for nontumorigenic cell lines. Here, we present the ability for human nontumorigenic microglial cells, which are the major immune cells in the central nervous system, to self-synthesize gold (Au) and iron (Fe) core nanoclusters, following exposures to metallic salts. We also show the ability for cells to internalize presynthesized Au and Fe mcNCs. Cellular fluorescence increased in most exposures and in a dose dependent manner in the case of Au salt. Scanning transmission electron microscopic imaging confirmed the presence of the metal within cells, while transmission electron microscopy images confirmed nanocluster structures and self-synthesis. Interestingly, self-synthesized nanoclusters were of similar size and internal structure as presynthesized mcNCs. Toxicity assessment of both salts and presynthesized NCs illustrated a lack of toxicity from Au salt and presynthesized NCs. However, Fe salt was generally more toxic and stressful to cells at similar concentrations.

A simple benzothiazole-based mitochondrial-targeting fluorescent probe for visualizing and monitoring viscosity in living cell, lung organ tissue, and living mice

The irregular cellular viscosity is associated with disease and malfunction at the cellular level, developing simple but effective fluorescence probe for detection of intracellular viscosity is of great significance. Herein, we report a benzothiazole-based fluorescent probe HBTD-V for sensitive response to viscosity change in biological systems. HBTD-V displays weak emission in low-viscosity media but strong fluorescence in high-viscosity media. Linear fitting of the viscosity titration results provided a good linear relationship between the fluorescence intensity (logI583) and viscosity (logη) within the viscosity range of 13.8–221.5 cp, indicating the capability of the probe for quantitative viscosity detection. Probe HBTD-V also holds some advantages such as excellent photostability, good anti-interference ability, and good mitochondrial targeting ability. Moreover, probe HBTD-V has been successfully employed to image the viscosity variations in living MCF-7 cells, mice lung organ and tissues, as well as in living mice body. The results demonstrate that HBTD-V can serve as a sensitive fluorescence probe for investigating the vital role of viscosity changes in complex biological systems.

Dual Targeting of Monomeric Tau and α-Synuclein Aggregation: A New Multitarget Therapeutic Strategy for Neurodegeneration.

Development of efficient multitargeted therapeutic strategies is crucial in facing the multifaceted nature of neurodegenerative diseases. Parkinson's disease (PD) and Alzheimer's disease (AD), the two most common neurodegenerative disorders, share a common hallmark of accumulation of misfolded protein aggregates which are Lewy bodies (LBs) and neurofibrillary tangles (NFTs), respectively. Tau protein and α-synuclein (α-syn), the precursors of LBs and NFTs, have demonstrated synergistic aggregation and neurotoxicity in both in vitro and in vivo models. Herein, we validate for the first time dual targeting of monomeric tau and α-syn aggregation as an efficient platform for development of multitarget therapeutics for neurological disorders. Cellular fluorescence resonance energy transfer (FRET)-based high-throughput screening for tau-binding compounds, followed by additional screening of the hits for their ability to impede α-syn aggregation identified MG-2119 as a potential lead. The high binding affinity of MG-2119 to monomeric tau was verified using cellular FRET assay, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and microscale thermophoresis (MSH). Moreover, MG-2119 inhibited α-syn aggregation as revealed by thioflavin T (ThT) assay and dynamic light scattering (DLS) measurements. Interestingly, MG-2119 was capable of rescuing combined tau and α-syn-induced cytotoxicity in SH-SY5Y neuroblastoma cells in a dose-dependent manner. Less pronounced cell-rescuing effects were observed for single-targeted tau and α-syn aggregation inhibitors showcasing the superiority of the multitargeted approach described in this study. The satisfactory pharmacokinetic profile and low toxicity of MG-2119 hold promise for future optimization to develop potential therapeutics for neurological disorders.

Bent Benzocoumarin Dyes that Fluoresce in Solution and in Solid State and Their Application to Bioimaging

AbstractOrganic fluorophores are essential in microscopic imaging of biological systems. New fluorophores with distinct features are in demand for bioimaging. Herein we report new bent benzocoumarin dyes that are fluorescent both in solution and in the solid state. The latter property enables us to form fluorescent nanoparticles with a mean size of 120–157 nm or larger depending on conditions. Some of the new dyes provide bright fluorescent images in cell, with formation of particulate spots that persist even after several washout procedures, and, furthermore, in tissue under two‐photon excitation at 900 nm. Such dyes have potential for the development of fluorescent probes for enhanced cellular fluorescence.

Reconstruction of Single-Cell Innate Fluorescence Signatures by Confocal Microscopy

Described here is confocal reflection microscopy-assisted single-cell innate fluorescence analysis (CRIF), a minimally invasive method for reconstructing the innate cellular fluorescence signature from each individual live cell in a population distributed in a three-dimensional (3D) space. The innate fluorescence signature of a cell is a collection of fluorescence signals emitted by various biomolecules within the cell. Previous studies established that innate fluorescence signatures reflect various cellular properties and differences in physiological status and are a rich source of information for cell characterization and identification. Innate fluorescence signatures have been traditionally analyzed at the population level, necessitating a clonal culture, but not at the single-cell level. CRIF is particularly suitable for studies that require 3D resolution and/or selective extraction of fluorescence signals from individual cells. Because the fluorescence signature is an innate property of a cell, CRIF is also suitable for tag-free prediction of the type and/or physiological status of intact and single cells. This method may be a powerful tool for streamlined cell analysis, where the phenotype of each single cell in a heterogenous population can be directly assessed by its autofluorescence signature under a microscope without cell tagging.

Proximal Tubule Epithelial Cell Reabsorption of the Drug Delivery Protein Elastin‐like Polypeptide

Elastin like polypeptide (ELP) is a protein composed of a pentapeptide repeat domain found in human tropoelastin. ELP is used for drug delivery by altering the ELP coding sequence to attach drugs, therapeutic peptides, or therapeutic proteins. One of the focuses of our group has been development of ELP – delivered therapeutics for treatment of kidney disease. Thus, understanding how ELP is filtered, reabsorbed, and distributed within the kidney is of key importance. In vivo studies in our lab showed that ELPs accumulated preferentially in the kidney after systemic administration. Renal deposition and intra‐renal distribution was dependent on ELP size, with smaller ELPs accumulating exclusively in the renal cortex and larger ELPs having a more mixed cortical and medullary distribution. All ELPs were present at high levels in tubular epithelial cells. However, whether the tubular distribution is the result of ELP reabsorption after filtration and whether the amount or rate of reabsorption is dependent on ELP size is unknown.To address this issue, uptake and intracellular distribution of fluorescein‐5‐malemide labeled ELPs of varying sizes were determined in primary human proximal tubule epithelial cells (HRPteC). HRPteC were seeded in a 6 well plate (4x104/well) and incubated with fluorescein labeled ELP (10μM) for various times (24hr, 6hr, 4hr, 2hr and 1hr). HRPteC were collected, and mean cellular fluorescence intensity was measured using flow cytometry. Percentage of ELP internalization was determined by quenching the extracellular fluorescein label with trypan blue (0.13% v/v), then repeating the flow cytometric analysis to measure only intracellular fluorescence. To obtain a qualitative representation of ELP interaction with kidney cells, HRPteC were seeded in an 8‐chamber glass slide (2x103 cells/chamber) and incubated with fluorescein labeled ELP (10μM) for various times as above. Following fixation and staining with 4′,6‐diamidino‐2‐phenylindole (DAPI), cells were imaged by laser scanning confocal microscopy. Our data demonstrate that a longer incubation time led to a greater accumulation of ELP in the proximal tubule epithelial cells. Analysis of internalization rate revealed that all ELPs entered the cells slowly over the course of a 24‐hour time frame. This finding was supported by confocal microscopy. Between 1 and 12 hours, localization of ELP occurred at the cellular membrane of the proximal tubule cells, but at time points between 12 and 24 hours, ELP was visible in punctate cytoplasmic structures likely to be endocytic vesicles. A smaller ELP‐63 (~25 kDa) accumulated in cells at higher levels than did a larger ELP‐159 (~62 kDa) after 24 hours of incubation (390.7 ±5.0 versus 651.6 ±42.7 RFU; p<0.05). These data suggest that there may be a common mechanism for tubular epithelial cell uptake of ELPs of various sizes, but that smaller ELPs may be internalized to a greater extent than larger ELPs. The results of these and future studies to determine the exact renal handling of ELPs of various sizes will be crucial to the design of ELPs for delivery of therapeutics to specific renal compartments or cell types.

Omentin-1 Ameliorated Free Fatty Acid-Induced Impairment in Proliferation, Migration, and Inflammatory States of HUVECs.

Objectives Endothelial cell injury is a critical pathological change during the development of atherosclerosis. Here, we explored the effect of omentin-1 on free fatty acid- (FFA-) induced endothelial cell injury. Methods An FFA-induced endothelial cell injury model was established to investigate the role of omentin-1 in this process. Cell proliferation was analyzed with the Cell Counting Kit assay and flow cytometry. Scratch and transwell assays were used to evaluate cell migration. Factors secreted by endothelial cells after injury were detected by western blotting, reverse-transcription quantitative polymerase chain reaction, and cellular fluorescence assay. Results Omentin-1 rescued the FFA-induced impaired proliferation and migration capabilities of human umbilical vein endothelial cells (HUVECs). It decreased the number of THP-1 cells attached to HUVECs in response to injury and inhibited the FFA-induced proinflammatory state of HUVECs. Conclusion Omentin-1 could partly ameliorate FFA-induced endothelial cell injury.

Facile preparation of FITC-modified silicon nanodots for ratiometric pH sensing and imaging.

A ratiometric fluorescent pH sensor was facilely constructed by covalent modification of amino-terminated silicon nanodots (SiND) with pH-sensitive fluorescein isothiocyanate (FITC). After optimization, the SiND-FITC(40:1) material with a SiND:FITC initial mass ratio of 40:1 was selected for the sensing of hydrogen ions. It was observed that the material inherits the unique features of SiND and FITC, and there is significant improvement of SiND acid-base stability, which is a favorable factor in terms of providing fluorescence reference signal. The SiND-FITC(40:1) material displays not only high pH sensitivity, but also good stability and anti-interference ability, and the response process is highly reversible. Deploying the SiND-FITC(40:1) material, we have made available a simple, sensitive, and precise approach for pH sensing. In aqueous solutions, the I517/I466 fluorescence intensity ratio of SiND-FITC(40:1) increases linearly in the pH range of 5.40-7.76. This dual emission nanosensor was successfully applied for pH sensing and cellular fluorescence imaging.

Ultra-fast Cycling for Multiplexed Cellular Fluorescence Imaging.

Rapid analysis of single and scant cell populations is essential in modern diagnostics, yet existing methods are often limited and slow. Herein, we describe an ultra-fast, highly efficient cycling method for the analysis of single cells based on unique linkers for tetrazine (Tz)/trans-cyclooctene (TCO)-mediated quenching. Surprisingly, the quenching reaction rates were more than 3 orders of magnitude faster (t1/2 <1 s) than predicted. This allowed multi-cycle staining and immune cell profiling within an hour, leveraging the accelerated kinetics to open new diagnostic possibilities for rapid cellular analyses.

Ultra-fast cycling for multiplexed cellular fluorescence imaging.

Rapid analysis of single and scant cell populations is essential in modern diagnostics, yet existing methods are often limited and slow. Here we describe an ultra-fast, highly efficient cycling method for the analysis of single cells based on unique linkers for tetrazine (Tz) / trans-cyclooctene (TCO) mediated quenching. Surprisingly, the quenching reaction rates were more than 3 orders of magnitude faster (t1/2 < 1 sec) than predicted. This allowed multi-cycle staining and immune cell profiling within an hour, leveraging the accelerated kinetics to open new diagnostic possibilities for rapid cellular analyses.

A fluorescent turn-on probe for detection of hypochlorus acid and its bioimaging in living cells

Hypochlorous acid has played several functions in the biological system. However, excess HOCl can cause damage to biomolecules and result in some diseases. Accordingly, a new fluorescent probe, BSP, has been developed for fast recognition of HOCl through the HOCl-induced oxidation of methyl phenyl sulfide to sulfoxide. The reaction of BSP with HOCl caused a 22-fold fluorescence enhancement (quantum yield increase from 0.006 to 0.133). The detection limit of HOCl is found to be 30nM (S/N=3). The fluorescence enhancement is due to the suppression of the photo-induced electron transfer from the methyl phenyl sulfide moiety to BODIPY. Eventually, the cellular fluorescence imaging experiment showed that BSP could be effectively used for monitoring HOCl in living cells.

Arsenene: A Potential Therapeutic Agent for Acute Promyelocytic Leukaemia Cells by Acting on Nuclear Proteins.

Arsenene has recently emerged as a promising new two-dimensional material for biomedical applications because of its excellent optical and electronic properties. Herein, novel 2D arsenene nanosheets were synthesized and shown to be effective against NB4 promyelocytic leukaemia (APL) cells (82 % inhibition) as well as inducing apoptosis while showing no toxicity towards normal cells. The high zeta potential, small size, and the planar structure were crucial to the toxicity of the materials. Label-free proteomic profiling analysis suggested that arsenene affected nuclear DNA replication, nucleotide excision repair, and pyrimidine metabolism pathways by downregulating the DNA polymerases POLE, POLD1, POLD2, and POLD3. Mass spectrometric studies showed that arsenene bound mainly to nuclear nucleotide acid binding proteins in NB4 cells and further cellular fluorescence studies revealed that the arsenene destroyed the nuclei. In vivo toxicity tests in mice also indicated the physiological biosafety of arsenene.

Arsenene: A Potential Therapeutic Agent for Acute Promyelocytic Leukaemia Cells by Acting on Nuclear Proteins

AbstractArsenene has recently emerged as a promising new two‐dimensional material for biomedical applications because of its excellent optical and electronic properties. Herein, novel 2D arsenene nanosheets were synthesized and shown to be effective against NB4 promyelocytic leukaemia (APL) cells (82 % inhibition) as well as inducing apoptosis while showing no toxicity towards normal cells. The high zeta potential, small size, and the planar structure were crucial to the toxicity of the materials. Label‐free proteomic profiling analysis suggested that arsenene affected nuclear DNA replication, nucleotide excision repair, and pyrimidine metabolism pathways by downregulating the DNA polymerases POLE, POLD1, POLD2, and POLD3. Mass spectrometric studies showed that arsenene bound mainly to nuclear nucleotide acid binding proteins in NB4 cells and further cellular fluorescence studies revealed that the arsenene destroyed the nuclei. In vivo toxicity tests in mice also indicated the physiological biosafety of arsenene.

Adenomyosis-derived extracellular vesicles endow endometrial epithelial cells with an invasive phenotype through epithelial-mesenchymal transition

Extracellular vesicles from highly metastatic tumor cells have been shown to mediate epithelial-mesenchymal transition (EMT)-related events in recipient cells. In endometrial epithelial cells, EMT processes are known to be involved in the development of adenomyosis. We aimed to investigate whether adenomyosis-derived extracellular vesicles (AMEVs) are able to induce an EMT process in endometrial epithelial cells. In this study, AMEVs were isolated from patients with adenomyosis and characterized by transmission electron microscopy, Western blot, and nanoparticle tracking. Primary endometrial epithelial cells (EECs) were derived from normal endometrium tissues from patients with leiomyoma and co-cultured with AMEVs in vitro. AMEV uptake was examined by fluorescence confocal microscopy. The invasion of EECs was confirmed by Transwell assay. Immunohistochemistry, Western blot, and qRT-PCR were performed on EECs to illustrate the expression levels of cytokeratin 19, E-cadherin, vimentin, and zinc finger E-box-binding homeobox 1 (ZEB1). The results indicated that the cellular fluorescence intensity gradually increased after 48 h of co-culture, but decreased after 72 h. After co-culturing with AMEVs for 72 h, EECs expressed significantly lower levels of cytokeratin 19 and E-cadherin, and significantly higher levels of vimentin and ZEB1. Together these results demonstrated that AMEVs induce an EMT process and enhance the invasion of EECs. These changes may contribute to the pathogenesis and progression of adenomyosis.

Flow Cytometry Measurement of Reactive Oxygen Species Generation: Some Caveats and Their Solution

The redox state is very important in normal physiology and pathology. Yet, its measurement is not practiced within the clinic. The explanation is principally methodological—the inadequacy of the methods for application in the clinical laboratory. Flow cytometry is routinely utilized in hemato-oncology and immunology. Measuring of the cellular fluorescence by flow cytometry following loading with 2′7′-dichlorodihydrofluorescin diacetate (DCFDA) has become a standard experimental method for quantification of reactive oxygen species (ROS). However, the DCF fluorescence depends on the probe uptake and esterification as well as the presence of intracellular quenchers. Cells of various types, stages of differentiation or senescence, or under different conditions (e.g., pathological vs. normal) may differ in these respects. A one-time measurement does not take into consideration these differences. Herein, we describe a protocol that overcomes these caveats by determining the change in DCF fluorescence with time as a measure of the rate of ROS generation. Normal (n = 20) and thalassemia (n = 20) red blood cells (RBCs) samples were loaded with DCFDA, washed, and then incubated in a DCFDA-free medium. DCF fluorescence was measured at different times of incubation by flow cytometry and the mean fluorescence (MFC) calculated. The results showed that the rate of increase in the DCF fluorescence was linear in both types of RBCs, regardless of their basal level and hemoglobin content, with thalassemia RBCs demonstrating a 4.5-fold higher rate (265/h) compared with normal RBCs (58/h) (p < 0.001). In conclusion, this pulse-chase procedure may be used for flow cytometry clinical evaluation of general and cell type-specific oxidative stress under normal and pathological conditions.