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Flow cytometry and micro-Raman spectroscopy: Identification of hemocyte populations in the mussel Mytilus galloprovincialis (Bivalvia: Mytilidae) from Faro Lake and Tyrrhenian Sea (Sicily, Italy)
摘要： Immunological and structural characteristics of hemocyte populations in the mussel Mytilus galloprovincialis (Bivalvia: Mytilidae), going from two different Sicilian habitats (Faro Lake and Tyrrhenian sea), was investigated by means of two different techniques (flow cytometric and micro-Raman spectroscopy analyses). For this purpose, three hundred and sixty mussels Mytilus galloprovincialis were analyzed during November 2017. They were divided into two equal groups (triplicate sample) on the basis of the site of collection (n = 60 caught in Faro Lake - group A, and n = 60 caught in Tyrrhenian Sea - group B). Some several differences between the species of Faro Lake and Tyrrhenian Sea are observed and ascribed to the disruption of immune parameters induced by the variations of some qualitative water parameters (temperature, salinity, dissolved oxygen, pH, ammonium 10, free chlorine, total chlorine, total phosphate, orthofhosphate) recorded in the two habitats. This study is relevant for monitoring the conditions of the sea and Faro Lake, which is strongly influenced by the currents of the Tyrrhenian Sea. Faro lake is well known for the cultivation of mussels and this is part of a coastal habitat of particular interest, consisted of a peculiar biocenotic complex. Further, for the first time, significant different arrangement in the mussels cell structural organization was evidenced by simply following their highly reproducible Raman biomolecular signatures.
关键词： Hemocyte,Bivalve immunology,Deep-sea,Mussel Mytilus galloprovincialis,Flow cytometry,Raman spectroscopy
更新于2025-09-23 15:23:52
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Calibration and characterization of intracellular Asante Potassium Green probes, APG-2 and APG-4
摘要： The response of fluorescent ion probes to ions is affected by intracellular environment. To properly calibrate them, intracellular and extracellular concentrations of the measured ion must be made equal. In the first, computational, part of this work, we show, using the example of potassium, that the two requirements for ion equilibration are complete dissipation of membrane potential and high membrane permeability for both potassium and sodium. In the second part, we tested the ability of various ionophores to achieve potassium equilibration in Jurkat and U937 cells and found a combination of valinomycin, nigericin, gramicidin and ouabain to be the most effective. In the third part, we applied this protocol to two potassium probes, APG-4 and APG-2. APG-4 shows good sensitivity to potassium but its fluorescence is sensitive to cell volume. Because ionophores cause cell swelling, calibration buffers had to be supplemented with 50 mM sucrose to keep cell volume constant. With these precautions taken, the average potassium concentrations in U937 and Jurkat cells were measured at 132 mM and 118 mM, respectively. The other tested probe, APG-2, is nonselective for cations; this is, however, a potentially useful property because the sum [K+] + [Na+] determines the amount of intracellular water.
关键词： Calibration,Ionophores,DiBAC4(3),Intracellular potassium,Cell volume,Valinomycin,ION potassium green,Gramicidin,Asante potassium green,Flow cytometry
更新于2025-09-23 15:23:52
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Rapid Identification of Functional Pyrrolysyl-tRNA Synthetases via Fluorescence-Activated Cell Sorting
摘要： The orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair and their variants have provided powerful tools for expanding the genetic code to allow for engineering of proteins with augmented structure and function not present in Nature. To expedite the discovery of novel pyrrolysyl-tRNA synthetase (PylRS) variants that can charge non-natural amino acids into proteins site-specifically, herein we report a streamlined protocol for rapid construction of the pyrrolysyl-tRNA synthetase library, selection of the functional PylRS mutants using fluorescence-activated cell sorting, and subsequent validation of the selected PylRS mutants through direct expression of the fluorescent protein reporter using a single bacterial strain. We expect that this protocol should be generally applicable to rapid identification of the functional PylRS mutants for charging a wide range of non-natural amino acids into proteins.
关键词： non-natural amino acids,flow cytometry,mutagenesis,amber codon suppression,genetic code expansion
更新于2025-09-23 15:23:52
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Visualizing Interactions of Circulating Tumor Cell and Dendritic Cell in the Blood Circulation Using In Vivo Imaging Flow Cytometry
摘要： Objective: Visualizing cell interactions in blood circulation is of great importance in studies of anticancer immunotherapy or drugs. However, the lack of a suitable imaging system hampers progress in this field. Methods: In this work, we built a dual-channel in vivo imaging flow cytometer to visualize the interactions of circulating tumor cells (CTCs) and dendritic cells (DCs) simultaneously in the bloodstream. Two artificial neural networks were trained to identify blood vessels and cells in the acquired images. Results and Conclusion: Using this technique, single CTCs and CTC clusters were readily distinguished by their morphology. Interactions of CTCs and DCs were identified, while their moving velocities were analyzed. The CTC-DC clusters moved at a slower velocity than that of single CTCs or DCs. This may provide new insights into tumor metastasis and blood rheology. Significance: This in vivo imaging flow cytometry system holds great potential for assessing the efficiency of targeting CTCs with anticancer immune cells or drugs.
关键词： Cell Interaction,Circulating Tumor Cell,In Vivo Imaging Flow Cytometry,Artificial Neural Network,Dendritic Cell
更新于2025-09-23 15:22:29
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Flow Cytometry Identifies a Spectrum of Maturation in Myeloid Neoplasms Having Plasmacytoid Dendritic Cell Differentiation
摘要： Background: Neoplasms derived from plasmacytoid dendritic cells (PDCs) are currently divided into two broad categories: mature PDC proliferations associated with myeloid neoplasms (MPDMN) and blastic plasmacytoid dendritic cell neoplasm (BPDCN); only BPDCN is recognized in the WHO 2016 classification of hematopoietic neoplasms. We present seven patients with high grade myeloid neoplasms (MNs), mostly acute leukemias, having a spectrum of PDC differentiation and not fitting with MPDMN or BPDCN. Methods: We analyzed seven MN cases having increased myeloblasts and prominent CD56-negative PDC proliferations comprising 5–26% of bone marrow or blood cellularity as measured by flow cytometry. The cases included five acute myeloid leukemia (three FAB M4 subtype, two unclassified), one mixed phenotype acute leukemia, and one case of unclassified MN. Results: Six cases demonstrated immunophenotypic evidence of PDC differentiation from leukemic blasts, based on variable expression of CD34, CD45, CD123, and CD304 by the leukemic cells. Four cases had circulating PDC populations in blood. None of the cases met clinical or pathologic criteria for BPDCN. Morphologic review was available for four acute leukemia cases and demonstrated either nodular or interstitial infiltrates of PDCs. All cases had an aggressive clinical course, and three cases had FLT3 ITD mutation. Conclusions: These cases demonstrate that high grade MNs, in particular AML, can exhibit PDC differentiation, with or without monocytic differentiation, in a manner distinct from MPDMN or BPDCN. The existence of MNs with immature PDC proliferations suggests that there is a broader spectrum of PDC-associated neoplasms than currently recognized.
关键词： plasmacytoid dendritic cell,flow cytometry,acute myeloid leukemia,myeloid neoplasm,immunophenotype
更新于2025-09-23 15:22:29
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Stability of eosin-5'-maleimide dye used in flow cytometric analysis for red cell membrane disorders
摘要： The eosin-5'-maleimide (EMA) binding test using flow cytometry is a common method to measure reduced mean channel fluorescence (MCF) of EMA-labeled red blood cells (RBCs) from patients with red cell membrane disorders. The basic principle of the EMA-RBC binding test involves the covalent binding of EMA to lysine-430 on the first extracellular loop of band 3 protein. In the present study, the MCF of EMA was analyzed for samples derived from 12 healthy volunteers (controls) to determine the stability (i.e., the percentage decrease in fluorescence) of EMA over a period of 1 year. Comparison of periodical MCF readings over time, that is, at 2-month intervals, showed that there were no significant changes in mean channel fluorescence for up to 6 months; however, there was a significant decrease in MCF at 8 months. For optimal dye utilization, EMA remained stable only for up to 6 months. Therefore, we recommend reconstitution of the dye every 6 months when implementing this test and storage at -80oC in dark conditions.
关键词： Flow Cytometry,Red Blood cells,Hereditary spherocytosis
更新于2025-09-23 15:22:29
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Preparation and evaluation of fluorescent poly(p-phenyleneethylene) covalently coated microspheres with reactive sites for bioconjugation
摘要： Fluorescent microspheres with reactive sites for interacting with biomolecules are greatly demanded in flow cytometry based suspension array. Aiming to develop new method for preparing fluorescent microspheres, two poly(p-phenyleneethylene) (PPE) conjugated polymers (CPs) with pedant carboxylic groups were synthesized via Sonogarshira coupling and followed with hydrolysis of ester groups; then the conjugated polymers were immobilized onto monodispersed amino-modified porous poly(glycidylmethacrylate) (APGMA) microspheres via coupling reaction between carboxylic and amino groups to give APGMA-CP fluorescent microspheres. The fluorescent microspheres were found to have good photo- and thermal stability as well as negligible influence from rigorous washing. The emission was uniform all across the inner and surface of the spheres. To evaluate the effectiveness of bioconjugation on the fluorescent microspheres, fluorescein isothiocyanate isomer I (FITC) labeled bovine serum albumin (BSA) (BSA-FITC) was chosen as the representative biomolecule to react with the fluorescent microspheres to give APGMA-CP-BSA-FITC. In the flow cytometry study, fluorescence compensation between the V500 and FITC detectors (receiving signals from fluorophores excited by 405 nm and 488 nm, respectively), to remove the interference between the emission of FITC and CPs, was realized using singly-stained microspheres. Finally, APGMA-CP-BSA-FITC microspheres were found to be double positive for CP and FITC with very high percentage (>95%), suggesting the bioconjugation is very effective. This study provides a facile method for simultaneous introduction of fluorescence and reactive sites onto the microspheres, which is very promising to be used as general strategy for fabricating fluorescence microspheres for application in high-throughput technology.
关键词： Fluorescence,Bioconjugation,Flow Cytometry,Microsphere,Conjugated Polymer
更新于2025-09-23 15:22:29
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Using <i>Imaging</i> Flow Cytometry to Quantify and Optimize Giant Vesicle Production by Water-in-oil Emulsion Transfer Methods
摘要： Many biologists, biochemists, and biophysicists study giant vesicles, which have a diameter of >1 μm, owing to their ease of characterization using standard optical methods. More recently, there has been interest in using giant vesicles as model systems for living cells and for the construction of artificial cells. In fact, there have been a number of reports about functionalizing giant vesicles using membrane-bound pore proteins and encapsulating biochemical reactions. Among the various methods for preparing giant vesicles, the water-in-oil emulsion transfer method is particularly well established. However, the giant vesicles prepared by this method have complex and heterogeneous properties, such as particle size and membrane structure. Here we demonstrate the characterization of giant vesicles by imaging flow cytometry to provide quantitative and qualitative information about the vesicle products prepared by the water-in-oil emulsion transfer method. Through image-based analyses, several kinds of protocol by-products, such as oil droplets and vesicles encapsulating no target molecules, were identified and successfully quantified. Further, the optimal agitation conditions for the water-in-oil emulsion transfer method were found from detailed analysis of imaging flow cytometry data. Our results indicate that a sonication-based water-in-oil emulsion transfer method exhibited a higher efficiency in producing giant vesicles - about 10 times or higher than that of vortex- and rumble strip-based methods. It is anticipated that these approaches will be useful for fine-tuning giant vesicle production and subsequent applications.
关键词： water-in-oil emulsion transfer method,Giant vesicles,imaging flow cytometry,POPC
更新于2025-09-23 15:22:29
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Flow cytometric analysis of Xenopus laevis and X. tropicalis blood cells using acridine orange
摘要： Automated blood cell counters can distinguish cells based on their size and the presence or absence of a nucleus. However, most vertebrates have nucleated blood cells that cannot be counted automatically. We established an alternative automatic method for counting peripheral blood cells by staining cells with the fluorescent dye acridine orange (AO) and analysing cell populations using flow cytometry (FCM). As promising new animal models, we chose Xenopus laevis and three inbred strains of X. tropicalis. We compared the haematological phenotypes, including blood cell types, cell sizes, cellular structure, and erythrocyte lifespans/turnover rate among X. laevis and the three inbred strains of X. tropicalis. Each cell type from X. laevis was sorted according to six parameters: forward- and side-scattered light emission, AO red and green fluorescence intensity, and cellular red and green fluorescence. Remarkably, the erythrocyte count was the highest in the Golden line, suggesting that genetic factors were associated with the blood cells. Furthermore, immature erythrocytes in anaemic X. laevis could be separated from normal blood cells based on red fluorescence intensity. These results show that FCM with AO staining allows for an accurate analysis of peripheral blood cells from various species.
关键词： X. tropicalis,flow cytometry,acridine orange,blood cells,Xenopus laevis
更新于2025-09-23 15:21:21
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OMIP‐0XX – 28‐color flow cytometry panel to characterize B cells and myeloid cells
摘要： This 28-color ?ow cytometry panel focuses on B cells, dendritic cells, and monocytes and was optimized for cryopreserved peripheral blood mononuclear cells (PBMC). In addition to markers enabling the analysis of monocytes (CD14) and de?nition of subsets within B cells (CD10, CD19, CD20, CD21, CD27, IgD, IgM) and dendritic cells (CD1c, CD11c, CD123, CD141, HLA-DR), we included functional markers such as chemokine receptors (CXCR3, CXCR5), surface immunoglobulins (IgA, IgD, IgG, IgM), Fc receptors (CD16, CD23, CD32, CD64), inhibitory molecules (CD73, CD85j), the co-stimulatory molecule CD40, and cytokine receptors (IL-21R, BAFF-R, TACI), which enables in-depth characterization of B cells, dendritic cells, and monocytes.
关键词： 28-color panel,B cells,dendritic cells,monocytes,flow cytometry
更新于2025-09-23 15:19:57