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oe1(光电查) - 科学论文

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  • Photoacoustic and fluorescent effects in multilayer plasmon-dye interfaces

    摘要： Progress in understanding the cell biology and progression of disease depends on advanced imaging and labeling techniques. Here, we address this demand by exploring novel multilayered nanocomposites (MNCs) with plasmonic nanoparticles and adsorbing dyes in thin nonabsorbing shells as supercontrast multimodal photoacoustic (PA) and fluorescent agents in the near-infrared range. The proof of concept was performed with gold nanorods (GNRs) and indocyanine green (ICG) dispersed in a matrix of biodegradable polymers. We demonstrated synergetic PA effects in MNCs with the gold–ICG interface that could not be achieved with ICG and GNRs alone. We also observed ultrasharp PA and emission peaks that could be associated with nonlinear PA and spaser effects, respectively. Low-toxicity multimodal MNCs with unique plasmonic, thermal, and acoustic properties have the potential to make a breakthrough in PA flow cytometry and near-infrared spasers in vivo by using the synergetic interaction of plasmonic modes with a nearby absorbing medium.

    关键词： gold nanorods,fluorescence,in vivo flow cytometry,biocompatible polymers,photoacoustic effect,indocyanine green,multilayer composite,fluorescence quenching

    更新于2025-09-10 09:29:36

  • Detection of gold nanorods uptake by macrophages using scattering analyses combined with diffusion reflection measurements as a potential tool for in vivo atherosclerosis tracking

    摘要： In this study, we report a potential noninvasive technique for the detection of vulnerable plaques using scatter analyses with flow cytometry (FCM) method combined with the diffusion reflection (DR) method. The atherosclerotic plaques are commonly divided into two major categories: stable and vulnerable. The vulnerable plaques are rich with inflammatory cells, mostly macrophages (MΦ), which release enzymes that break down collagen in the cap. The detection method is based on uptake of gold nanorods (GNR) by MΦ. The GNR have unique optical properties that enable their detection using the FCM method, based on their scattering properties, and using the DR method, based on their unique absorption properties. This work demonstrates that after GNR labeling of MΦ, 1) the FCM scatter values increased up to 3.7-fold with arbitrary intensity values increasing from 1,110 to 4,100 and 2) the DR slope changed from an average slope of 0.196 (MΦ only) to an average slope of 0.827 (MΦ labeled with GNR) (P0.001 for both cases). The combination of FCM and DR measurements provides a potential novel, highly sensitive, and noninvasive method for the identification of atherosclerotic vulnerable plaques, aimed to develop a potential tool for in vivo tracking.

    关键词： flow cytometry,macrophages,gold nanoparticles,vulnerable plaques,noninvasive detection

    更新于2025-09-10 09:29:36

  • Development of a Novel Flow Cytometry-Based System for White Blood Cell Differential Counts: 10-color LeukoDiff

    摘要： Background: Flow cytometry (FCM) is commonly used to identify many cell populations. We developed a white blood cell (WBC) differential counting system for detecting abnormal cells using FCM incorporating 10 colors and 11 antibodies in a single tube, called “10-color LeukoDiff,” and evaluated its performance. Methods: Ninety-one EDTA-anti-coagulated peripheral blood samples from 76 patients were analyzed using 10-color LeukoDiff. We compared 10 color LeukoDiff results with the results of manual differential count (manual diff). WBCs were classified into 17 cell populations: neutrophils, total lymphocytes, T lymphocytes, B lymphocytes, CD5 and CD19 co-expressing lymphocytes, natural killer cells, total monocytes, 16+ monocytes, eosinophils, immature granulocytes, basophils, myeloblasts, B-blasts, T-blasts, myeloid antigen-positive B-blasts, CD19- plasma cells, and 19+ plasma cells. Results: The correlations between the 10-color LeukoDiff and manual diff results were strong (r > 0.9) for mature neutrophils, lymphocytes, eosinophils, immature granulocytes, and blasts and moderate for monocytes and basophils (r = 0.86 and 0.74, respectively). There was no discrepancy in blast detection between 10-color LeukoDiff and manual diff results. Furthermore, 10-color LeukoDiff could differentiate the lineage of the blasts and separately count chronic lymphocytic leukemic cells and multiple myeloma cells. Conclusions: The 10-color LeukoDiff provided an accurate and comprehensive WBC differential count. The most important ability of 10-color LeukoDiff is to detect blasts accurately. This system is clinically useful, especially for patients with hematologic diseases, such as acute leukemia, chronic lymphocytic leukemia, and multiple myeloma. Application of this system will improve the development of FCM gating strategy designs.

    关键词： 10-color LeukoDiff,Flow cytometry,Blasts,Immature granulocytes,Manual differential count

    更新于2025-09-10 09:29:36

  • Novel DRAQ5?/SYTOX? Blue Based Flow Cytometric Strategy to Identify and Characterize Stem Cells in Human Breast Milk

    摘要： Human breast milk could be an important stem cell source for the development of newborn and preterm infants, but quantitative data on the stem cell content in breast milk at various gestational stages are needed to determine the clinical value of breast milk as a source of stem cells. Breast milk also contains milk fat globules, lipid droplets of different sizes, debris and dead cells and these components hamper flow cytometry analysis of human breast milk samples. Here, we originally used standard protocols for flow cytometry to characterize cell populations in human breast milk but failed to discriminate between cells and noncellular components. We then applied a centrifugation protocol to separate cream and skim milk from the cell-containing pellet and used a novel staining protocol with DRAQ5? and SYTOX? blue dye as well as antibodies to characterize cells within the pellet fraction. Flow cytometry analysis identified viable DRAQ5?+/SYTOX? Blue? cells and determined the content of CD11b+ monocytes and TRA-1-81+ putative stem cells in human breast milk samples. Hence, we developed a novel and reliable flow cytometry based-approach to quantify subpopulation of cells in human breast milk with a high content of milk fat globules, lipid droplets, and particles. This approach will improve the identification and quantification of breast milk cells and allow standardizing the flow cytometry-based evaluation of the stem cell content.

    关键词： Nile red,viability,DRAQ5,flow cytometry,TRA-1-81,CD11b,SYTOX blue,human breast milk cells

    更新于2025-09-10 09:29:36

  • Flow Cytometry - Select Topics || Sperm Flow Cytometry: Beyond Human Fertilization and Embryo Development

    摘要： Male infertily is a contributing factor in up to 50% of all infertility cases, a solo cause in about 30% of them. Therefore, new and improved diagnostic methods that reduce operator variability regarding sperm defects that are not accesible by the conventional microscope scoring should be evaluated. Assisted reproductive technology (ART) has been involved in the description of alternative pathways in basic cellular functions. it is important to know that it is also related to the peri-implantatory processes that involve the sperm-oocyte interaction, cellular changes observed during fertilization, and the early and late embryo development. Several pathways have been involved at the early stages of human gametogenesis. The spermatozoon has demonstrated an intricate correlation during the fertilization process, as a transfected vector on genetic material, and as interacting with other inner components (RNAm, mitochondrial organelles, etc.). Spermatogenesis is affected by programmed death cell pathways from its packaging process through the elongated cytoplasmic structures during spermiogenesis. Flow cytometry (FC) has been an outstanding tool with the capability to select human gametes to achieve a better reproductive condition. It has been applied as a diagnostic and therapeutic tool allowing a measurable and objective selection and discrimination of spermatozoa from subfertile subjects. Using FC, we are able to know that early distribution of organelles such as mitochondria has an impact in embryo quality before genetic activation on the eight-cell stages occurs. This chapter will let the readers know the current knowledge on sperm fertilization and the relation between the embryo development and the offspring and all the tools now available for an early diagnosis and to identify therapeutic options with FC.

    关键词： apoptosis,flow cytometry,spermatozoa,sperm,DNA fragmentation,embryo development,fertilization

    更新于2025-09-09 09:28:46

  • Detecting intracellular thiol redox state in leukaemia and heterogeneous immune cell populations: An optimised protocol for digital flow cytometers

    摘要： Flow cytometric methods for detecting and quantifying reduced intracellular thiol content using fluorescein-5-maleimide (F5M) in viable eukaryotic cells date back to 1983 (Durand and Olive [1]). There has been little development in these methodologies since that time, a period that has witnessed huge technological advances, particularly with the emergence of digital multi-parameter flow cytometric systems. Concurrent advancement in our understanding of redox regulation within eukaryotic cellular systems has also followed, whereby it is now accepted that cysteine thiols partake in redox reactions, which regulate protein activity and function (Groitl and Jakob (2014), Won et al. (2012)). Moreover, we are at the dawn of a new era in redox biology whereby the importance of 'reductive stress' in eukaryotic cellular systems is gathering momentum (Wadley et al. (2018) [4]). It is therefore critical that methods be continually advanced to better understand these concepts in more detail at the cellular level. Flow cytometry is a powerful technique that may be used for this purpose. Henceforth we have rejuvenated these methods to address modern scientific questions. In this paper, essential detail is provided on: The adaption of a protocol initially described by Durand and Olive [1] for use with modern digital flow cytometer configurations. Here we provide optimal conditions for labelling intracellular thiols with F5M for detection using digital flow cytometers. Our modifications avoid the use of methanol fixation thus preserving cell viability in single cell suspension cultures. Demonstration that flow cytometry can detect the gain and loss of reduced intracellular thiols in cells exposed to physiological doses of hydrogen peroxide mediated by glucose oxidase (Hole et al. (2013) [5]). Validation of F5M protein labelling by coupling method to confocal microscopy and downstream proteomics, thus permitting a powerful experimental platform for potential use with next generation flow cytometry e.g. CyTOF (Lin and Maecker (2018) [6]).

    关键词： Fluorescein-5 maleimide,Reactive oxygen species,Oxidative stress,Reductive stress,Flow cytometry

    更新于2025-09-09 09:28:46

  • Flow cytometry reticulocyte counting using acridine orange: validation of a new protocol

    摘要： Introduction: Currently, the reticulocyte counting is a challenge for clinical laboratories in Brazil, mainly for the ordinary ones, which still use the manual method. This method has some limitations, since it consists of a laborious method, time consuming, with low accuracy. Objectives: This study has developed and evaluated the performance of a New Laboratory Protocol for flow cytometry (FC) reticulocytes counting using acridine orange (AO) as dye, aiming to standardize a more precise, easy, fast implementation, and low cost protocol. After standardization of the New Protocol (FC/AO), it was compared with the manual method. The results were analyzed according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS), now known as Clinical and Laboratory Standards Institute (CLSI), to evaluate the interchangeability of methods in linear regression analysis and paired t test, besides other quality control tests. Conclusion: Based on these results concerning to the correlation between the methods and the tests related to quality control, we can admit that FC/AO for reticulocyte counting shows undeniable advantages when compared to the preexisting manual method.

    关键词： flow cytometry,manual reticulocyte count.,reticulocyte counting methods,acridine orange

    更新于2025-09-04 15:30:14

  • Imaging flow cytometry and confocal microscopy-based examination of F-actin and phosphoinositide dynamics during leukocyte immune-type receptor-mediated phagocytic events

    摘要： Cells of the innate immune system rapidly detect and eliminate invading microbes using surface-expressed immunoregulatory receptors that translate extracellular binding events into potent effector responses. Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are a family of immunoregulatory proteins that have been shown to regulate several innate immune cell effector responses including the phagocytic process. The mechanisms by which these receptors regulate phagocytosis are not entirely understood but we have previously shown that different IpLITR-types use ITAM-dependent as well as ITAM-independent pathways for controlling target engulfment. The main objective of this study was to develop and use imaging flow cytometry and confocal microscopy-based assays to further examine both F-actin and phosphoinositide dynamics that occur during the different IpLITR-mediated phagocytic pathways. Results show that the ITAM-dependent IpLITR-induced phagocytic response promotes canonical changes in F-actin polymerization and PI(4,5)P2 redistributions. However, the ITAM-independent IpLITR phagocytic response induced unique patterns of F-actin and PI(4,5)P2 redistributions, which are likely due to its ability to regulate alternative signaling pathways. Additionally, both IpLITR-induced phagocytic pathways induced target internalization into PI(3)P-enriched phagosomes indicative of a maturing phagosome compartment. Overall, this imaging-based platform can be further applied to monitor the recruitment and distribution of signaling molecules during IpLITR-mediated phagocytic processes and may serve as a useful strategy for functional examinations of other immunoregulatory receptor-types in fish.

    关键词： Cytoskeletal dynamics,Teleost,Phosphoinositides,Leukocyte immune-type receptors,Imaging,Phagocytosis,Innate immunity,Fluorescent probes,F-actin,Flow cytometry

    更新于2025-09-04 15:30:14

  • Flow cytometry for fast screening and automated risk assessment in systemic light-chain amyloidosis

    摘要： Early diagnosis and risk stratification are key to improve outcomes in light-chain (AL) amyloidosis. Here we used multidimensional-flow-cytometry (MFC) to characterize bone marrow (BM) plasma cells (PCs) from a series of 166 patients including newly-diagnosed AL amyloidosis (N = 94), MGUS (N = 20) and multiple myeloma (MM, N = 52) vs. healthy adults (N = 30). MFC detected clonality in virtually all AL amyloidosis (99%) patients. Furthermore, we developed an automated risk-stratification system based on BMPCs features, with independent prognostic impact on progression-free and overall survival of AL amyloidosis patients (hazard ratio: ≥ 2.9;P ≤ .03). Simultaneous assessment of the clonal PCs immunophenotypic protein expression profile and the BM cellular composition, mapped AL amyloidosis in the crossroad between MGUS and MM; however, lack of homogenously-positive CD56 expression, reduction of B-cell precursors and a predominantly-clonal PC compartment in the absence of an MM-like tumor PC expansion, emerged as hallmarks of AL amyloidosis (ROC-AUC = 0.74;P < .001), and might potentially be used as biomarkers for the identification of MGUS and MM patients, who are candidates for monitoring pre-symptomatic organ damage related to AL amyloidosis. Altogether, this study addressed the need for consensus on how to use flow cytometry in AL amyloidosis, and proposes a standardized MFC-based automated risk classification ready for implementation in clinical practice.

    关键词： bone marrow,plasma cells,light-chain amyloidosis,risk assessment,flow cytometry

    更新于2025-09-04 15:30:14

  • A Flow Cytometry‐Based Assay for High‐Throughput Detection and Quantification of Neutrophil Extracellular Traps in Mixed Cell Populations

    摘要： Neutrophil extracellular traps (NETs) are web-like structures composed of decondensed chromatin and antimicrobial proteins that are released into the extracellular space during microbial infections. This active cell death program is known as NETosis. To date, fluorescence microscopy is the widely accepted method for visualization and quantification of NETs. However, this method is subjective, time consuming and yields low numbers of analyzed polymorphonuclear cells (PMNs) per sample. Increasing interest has emerged on the identification of NETs using flow cytometry techniques. However, flow cytometry analysis of NETs requires particular precautions for sample preparation to obtain reproducible data. Herein, we describe a flow cytometry-based assay for high-throughput detection and quantification of NETosis in mixed cell populations. We used fluorescent-labeled antibodies against cell markers on PMNs together with a combination of nucleic acid stains to measure NETosis in whole blood (WB) and purified PMNs. Using plasma membrane-impermeable DNA-binding dye, SYTOX Orange (SO), we found that cell-appendant DNA of NETting PMNs were positive for SO and DAPI. The combination of optimally diluted antibody and nucleic acid dyes required no washing and yielded low background fluorescence. Significant correlations were found for NETosis from WB and purified PMNs. We then validated the assay by comparing with time-lapse live cell fluorescence microscopy and determined very good intraassay and interassay variances. The assay was then applied to a disease associated with NETosis, systemic lupus erythematosus (SLE). We examined PMA-induced NETosis in peripheral PMNs from SLE patients and controls and in bone marrow PMNs from multiple murine models. In summary, this assay is observer-independent and allows for rapid assessment of a large number of PMNs per sample. Use of this assay does not require sophisticated microscopic equipment like imaging flow cytometers and may be a starting point to analyze extracellular trap formation from immune cells other than PMNs.

    关键词： neutrophils,flow cytometry,extracellular traps

    更新于2025-09-04 15:30:14