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High-throughput label-free molecular fingerprinting flow cytometry
摘要： Flow cytometry is an indispensable tool in biology for counting and analyzing single cells in large heterogeneous populations. However, it predominantly relies on fluorescent labeling to differentiate cells and, hence, comes with several fundamental drawbacks. Here, we present a high-throughput Raman flow cytometer on a microfluidic chip that chemically probes single live cells in a label-free manner. It is based on a rapid-scan Fourier-transform coherent anti-Stokes Raman scattering spectrometer as an optical interrogator, enabling us to obtain the broadband molecular vibrational spectrum of every single cell in the fingerprint region (400 to 1600 cm?1) with a record-high throughput of ~2000 events/s. As a practical application of the method not feasible with conventional flow cytometry, we demonstrate high-throughput label-free single-cell analysis of the astaxanthin productivity and photosynthetic dynamics of Haematococcus lacustris.
关键词： high-throughput,astaxanthin,label-free,single-cell analysis,microfluidics,flow cytometry,Raman spectroscopy,Haematococcus lacustris,coherent anti-Stokes Raman scattering
更新于2025-09-19 17:15:36
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Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells
摘要： T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.
关键词： lysosome,Flow cytometry,organelle-specific dyes,mitochondria,multicolor,T cell
更新于2025-09-19 17:15:36
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A novel aspect of functionalized graphene quantum dots in cytotoxicity studies
摘要： Graphene quantum dots (GQDs) represent a new generation of graphene-based nanomaterials with enormous potential for use and development of a variety of biomedical applications. However, up to now little studies have investigated the impact of GQDs on human health in case of exposure. GQDs were synthesized from citric acid as carbon precursor by hydrothermal treatment at 160 °C for 4 h. The synthesized GQDs showed strong blue emission under UV-Irradiation with fluorescence quantum yield of 9.8%. The obtained GQDs were further functionalized by nitric acid vapor method. Nitrogen carbonized, activated and adsorption/desorption isotherms were used to analyze the surface area and porous structures of GQDs. The results revealed that compared to GQDs, the specific surface area of functionalized graphene quantum dots (fGQDs) has been increased from 0.0667 to 2.5747 m2/g and pore structures have been enhanced significantly. The potential cytotoxic effect of GQDs, fGQDs and GO suspensions was evaluated on HFF cell line using MTT assays and flow cytometry method after 24 h incubation. We have for the first time demonstrated that by carbonization, activation and functionalization of GQDs they still showed cytocompatible properties. We observed excellent biocompatibility of GQDs and fGQDs at low concentrations. Moreover, the results suggested that modification of GQDs yields product suspensions with high surface area, enhanced pore volume and loading capacities. Thus, fGQDs represent an attractive candidate for further use in drug delivery systems and bio-imaging application.
关键词： Graphene quantum dot,Biocompatibility,Cytotoxicity,Flow cytometry,Functionalized graphene quantum dot
更新于2025-09-19 17:13:59
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Investigation of Cell Growth and Chlorophyll a Content of the Coccolithophorid Alga Emiliania huxleyi by Using Simple Bench-Top Flow Cytometry
摘要： The coccolithophorid alga Emiliania huxleyi produces micro-structured calcite particles, which are called coccoliths. Due to their unique and sophisticated structure, coccoliths are highly promising for different industrial applications, such as paper manufacturing, color and lacquer preparation. The mass production of coccoliths requires the evaluation of optimum cultivation conditions. This study investigates the impact of varying irradiance (10-1500 μmol m-2 s-1) on growth and chlorophyll a content of two calcifying strains CCMP371 and RCC1216 as well as on the non-calcifying strain RCC1217 (haploid form of RCC1217). The light kinetics contradicts the popular opinion, that E. huxleyi is an extraordinarily light tolerating alga in general. Photoinhibition was already observed at irradiance >500 μmol m-2 s-1 in the case of the calcifying strains. Furthermore, light requirements to grow at maximum growth rate, as well as thresholds towards photoinhibition were considerably different between calcifying and non-calcifying strains. The haplont required significantly higher irradiance to reach maximum μspec (>200 μmol m-2 s-1), while being much more tolerant to towards photoinhibition, which occurred not until 800 μmol m-2 s-1. Furthermore, a novel method was proposed to allow for the estimation of chlorophyll a content from flow cytometry data. By comprising an Advanced Fluorescence Ratio (AFLR), which considers culture heterogeneity, this method enables for simple chlorophyll a estimation also in older cultures of calcifying Emiliania huxleyi, which tend to build agglomerates.
关键词： Autofluorescence,characterization,Photosynthetic pigments,Flow cytometry,Chlorophyll,Microalgae
更新于2025-09-16 10:30:52
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A Model for the Binding of Fluorescently Labeled Anti-Human CD4 Monoclonal Antibodies to CD4 Receptors on Human Lymphocytes
摘要： The CD4 glycoprotein is a component of the T cell receptor complex which plays an important role in the human immune response. This manuscript describes the measurement and modeling of the binding of fluorescently labeled anti-human CD4 monoclonal antibodies (mAb; SK3 clone) to CD4 receptors on the surface of human peripheral blood mononuclear cells (PBMC). CD4 mAb fluorescein isothiocyanate (FITC) and CD4 mAb allophycoerythrin (APC) conjugates were obtained from commercial sources. Four binding conditions were performed, each with the same PBMC sample and different CD4 mAb conjugate. Each binding condition consisted of the PBMC sample incubated for 30 min in labeling solutions containing progressively larger concentrations of the CD4 mAb-label conjugate. After the incubation period, the cells were re-suspended in PBS-based buffer and analyzed using a flow cytometer to measure the mean fluorescence intensity (MFI) of the labeled cell populations. A model was developed to estimate the equilibrium concentration of bound CD4 mAb-label conjugates to CD4 receptors on PBMC. A set of parameters was obtained from the best fit of the model to the measured MFI data and the known number of CD4 receptors on PBMC surface. Divalent and monovalent binding had to be invoked for the APC and FITC CD4 mAb conjugates, respectively. This suggests that the mAb binding depends on the size of the label, which has significant implications for quantitative flow cytometry. The study supports the National Institute of Standards and Technology program to develop quantitative flow cytometry measurements.
关键词： fluorescence spectroscopy,PBMC,FITC,antibody binding,CD4 monoclonal antibody,flow cytometry,APC,cooperative binding
更新于2025-09-12 10:27:22
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Magneto-fluorescent microbeads for bacteria detection constructed from superparamagnetic Fe <sub/>3</sub> O <sub/>4</sub> nanoparticles and AIS/ZnS quantum dots
摘要： The efficient and sensitive detection of pathogenic microorganisms in aqueous environments such as water used in medical applications, drinking water, and cooling water of industrial plants requires simple and fast methods suitable for multiplexed detection such as flow cytometry (FCM) with optically encoded carrier beads. For this purpose, we combine fluorescent Cd-free Ag-In-S ternary quantum dots (t-QDs) with fluorescence lifetimes (LTs) of several hundred nanoseconds and superparamagnetic Fe3O4 nanoparticles (SPIONs) with mesoporous CaCO3 microbeads to a magneto-fluorescent bead platform that can be surface-functionalized with bioligands such as antibodies. This inorganic bead platform enables immuno-magnetic separation, target enrichment, and target quantification with optical readout. The beads can be detected with steady-state and time-resolved fluorescence microscopy and flow cytometry (FCM). Moreover, they are suited for readout by time gated emission. In the following, the preparation of these magneto-fluorescent CaCO3 beads, their spectroscopic and analytic characterization, and their conjugation with bacteria-specific antibodies are presented as well as proof-of-concept measurements with Legionella pneumophila including cell cultivation and plating experiments for bacteria quantification. Additionally, the possibility to discriminate between the long-lived emission of the LT-encoded capture and carrier CaCO3 beads and the short-lived emission of the dye-stained bacteria with time-resolved fluorescence techniques and single wavelength excitation is demonstrated.
关键词： AIS/ZnS quantum dots,flow cytometry,magneto-fluorescent microbeads,time-resolved fluorescence,superparamagnetic Fe3O4 nanoparticles,bacteria detection
更新于2025-09-11 14:15:04
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Simplified Dynabeads Method Using Light Microscopy for Enumerating TCD4+ Lymphocytes in Resource-Limited Settings
摘要： Background: We demonstrated feasibility of implanting the Dynabeads method for CD4+ T lymphocyte enumeration in resource-poor settings (ANRS 1226 study). However, as this technique requires a fluorescence microscope which is not usually available in these settings, WHO has encouraged to simplify the method allowing TCD4+ lymphocyte counting under a light microscope. Methods: TCD4+ lymphocytes enumeration was assessed using Dynabeads after staining cells nuclei with non-fluorescent dyes and readings under light microscope (DLM). A total of 305 triple of values of CD4 cells counts were generated by both Dynabeads method using a light microscopy (DLM), Dynabeads method using a fluorescent microscope (DFM) and the single-platform flow cytometry technique (FCM). The accuracy of DLM was analyzed using 4 fresh blood samples showing 200, 400, 500 and 1000 cells/μl in FCM respectively. Correlations have been studied between the 3 methods. The DLM was then evaluated for its ability to correctly segregate absolute TCD4+ lymphocyte values at the thresholds of 200 cells/μl and 350 cells/μl. Findings: Cells nuclei staining with Sternheimer-Malbin, Turck1, and Giemsa allows TCD4+ lymphocytes enumeration using DLM. FCM has shown the greatest standard deviations and amplitudes. The reproducibility of DLM was better than FCM. The correlation coefficient between FCM and DFM was 0.975 and it was 0.973, 0.972 and 0.969 with DLM using Sternheimer-Malbin, Turck1 and Giemsa, respectively. The ability of DLM to correctly segregate TCD4+ lymphocyte values at the threshold of 200 cells/μl and 350 cells/μl was good. Conclusion: Reliable TCD4+ enumeration can be obtained with DLM. These results will contribute in resource-limited-settings to further reduce the cost of TCD4+ lymphocytes counting and make it more widely available in peripheral laboratories and even in central laboratories that face problems with maintenance and stock-out of reagents for flow cytometers.
关键词： Dynabeads method,Flow Cytometry,Resource-limited settings,Fluorescence microscopy,Light microscopy,TCD4+ lymphocytes enumeration
更新于2025-09-11 14:15:04
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Luminescence lifetime encoding in time-domain flow cytometry
摘要： Time-resolved flow cytometry represents an alternative to commonly applied spectral or intensity multiplexing in bioanalytics. At present, the vast majority of the reports on this topic focuses on phase-domain techniques and specific applications. In this report, we present a flow cytometry platform with time-resolved detection based on a compact setup and straightforward time-domain measurements utilizing lifetime-encoded beads with lifetimes in the nanosecond range. We provide general assessment of time-domain flow cytometry and discuss the concept of this platform to address achievable resolution limits, data analysis, and requirements on suitable encoding dyes. Experimental data are complemented by numerical calculations on photon count numbers and impact of noise and measurement time on the obtained lifetime values.
关键词： lifetime-encoded beads,multiplexing,Time-resolved flow cytometry,bioanalytics,time-domain measurements
更新于2025-09-11 14:15:04
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MicroRNA Quantitation During Dendritic Cell Endocytosis Using Imaging Flow Cytometry: Key Factors and Requirements
摘要： Background/Aims: MicroRNA (miRNA)-induced suppression of dendritic cells (DCs) has been implicated in many diseases. Therefore, accurate monitoring of miRNA endocytosis by DCs is important for understanding the role of miRNAs in many diseases. Recently, a method for measuring the co-localization of Argonaute 2 (AGO2)-associated miRNAs on laser-scanning confocal microscopy method was proposed to localize the miRNAs. But its definition was limited by the number of observed cells through its accuracy. Methods: In this study, a method based on imaging flow cytometry was developed to localize miR-590-5p with fluorescent probes in DCs. miR-590-5p proven to play an important role in tumor immunity. This method enabled the quantification, visualization and localization of the fluorescence intensity in 30,000 individual cells. Results: Using this method, the DCs with different endocytotic ability were distinguished. The behaviour of miR-590-5p during endocytosis under the stimulation of tumor antigen in DCs was observed, binding to its cognate target mRNA and degradation in DCs. Conclusion: This method based on imaging flow cytometry provide an additional method to study miRNA processing in DCs, which makes it a valuable addition to existing miRNA research techniques
关键词： miR-590-5p,miRNA endocytosis,Flow cytometry,Dendritic cells,Argonaute 2 (AGO2),FRET analysis
更新于2025-09-10 09:29:36
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Detection of Apoptotic Circulating Tumor Cells Using in vivo Fluorescence Flow Cytometry
摘要： Most cancer patients die from metastatic disease as a result of a circulating tumor cell (CTC) spreading from a primary tumor through the blood circulation to distant organs. Many studies have demonstrated the tremendous potential of using CTC counts as prognostic markers of metastatic development and therapeutic efficacy. However, it is only the viable CTCs capable of surviving in the blood circulation that can create distant metastasis. To date, little progress has been made in understanding what proportion of CTCs is viable and what proportion is in an apoptotic state. Here, we introduce a novel approach toward in situ characterization of CTC apoptosis status using a multi-color in vivo flow cytometry platform with fluorescent detection for the real-time identification and enumeration of such cells directly in blood flow. The proof of concept was demonstrated with two-color fluorescence flow cytometry (FFC) using breast cancer cells MDA-MB-231 expressing green fluorescein protein (GFP), staurosporine as an activator of apoptosis, Annexin-V apoptotic kit with orange dye color, and a mouse model. The future application of this new platform for real-time monitoring of antitumor drug efficiency is discussed.
关键词： fluorescence flow cytometry,circulating tumor cells,in vivo detection of circulating tumor cells,apoptosis
更新于2025-09-10 09:29:36