Application Of Flow Cytometry Research Articles

Implementation and validation of MICaFVi: A highly efficient nanotechnology-based method for coronaviruses detection

Coronaviruses have emerged as a significant public health concern due to the global impact of Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which cause COVID-19. The development of sensitive and accurate detection methods is critical for early diagnosis, disease management, and outbreak control. In a previous study, we developed and optimized a nanobased detection methodology called Magnetic Immuno-Capture Followed by Flow Virometry (MICaFVi) using virus-mimicking silica nanoparticles and MERS-CoV/SARS-CoV-2 pseudoviral particles. In the present study, we have extended this methodology to evaluate its specificity and sensitivity for detecting wild-type MERS-CoV and SARS-CoV-2 in human and camel samples. Our results demonstrated that MICaFVi successfully detected MERS-CoV and SARS-CoV-2 viruses with high sensitivity and specificity, although it showed reduced performance for samples with Ct values of 30 or lower compared to qPCR. Despite some limitations in detection speed and sensitivity, MICaFVi represents a significant advancement in diagnostic methodologies by combining nanotechnology with flow cytometry. Additionally, we adapted the MICaFVi methodology to simultaneously detect MERS-CoV and SARS-CoV-2 in a single multiplex assay. The successful implementation of this advanced detection approach has important implications for improving early detection, surveillance, and control of both current and future viral or bacterial pandemics. Our results underscore the potential of MICaFVi as a valuable tool for monitoring the spread of these viruses and highlight its role in advancing diagnostic technologies. This extension of our earlier work offers new insights into the application of nanotechnology and flow cytometry for viral detection.

Using Spectral Flow Cytometry for CAR T-Cell Clinical Trials: Game Changing Technologies Enabling Novel Therapies

Monitoring chimeric antigen redirected (CAR) T-cells post-infusion in clinical trials is a specialized application of flow cytometry. Unlike the CAR T-cell monitoring for individual patients conducted in clinical laboratories, the data generated during a clinical trial will be used not only to monitor the therapeutic response of a single patient, but determine the success of the therapy itself, or even of an entire class of therapeutic compounds. The data, typically acquired at multiple testing laboratories, will be compiled into a single database. The data may also be used for mathematical modeling of cellular kinetics or to identify predictive biomarkers. With the expanded context of use, a robust, standardized assay is mandatory in order to generate a valuable and reliable data set. Hence, the requirements for assay validation, traceable calibration, technology transfer, cross-instrument standardization and regulatory compliance are high.

Applications of Flow Cytometry in Inborn Errors of Immunity

The Article Abstract is not available.

Application of Flow Cytometry for Viability Assessment of Mutants for Translation Termination Factors in the Yeast Saccharomyces cerevisiae

Nonsense mutations in the essential SUP45 and SUP35 genes, encoding translation termination factors, affect the viability of Saccharomyces cerevisiae cells. Flow cytometry revealed that the viability of mutants was 3.5‒4 times lower compared to the wild-type. Moreover, the mutants were found to have higher sensitivity to ultrasonic treatment.

Application of Online Flow Cytometry for Early Biofouling Detection in Reverse Osmosis Membrane Systems.

Biofouling poses a significant challenge to reverse osmosis (RO) membrane systems, necessitating timely detection for effective control. This study evaluated the efficacy of flow cytometry (FCM) for early biofilm detection in comparison to conventional system performance indicators. Feed channel pressure drop and total cell concentration in the Membrane Fouling Simulator (MFS) flowcell cross-flow outlet water were monitored over time as early biofouling indicators. The results demonstrated the potential of increased bacterial cell concentration in cross-flow outlet water as a reliable indicator of biofouling development on the membrane. Water outlet monitoring enabled faster biofouling detection compared to feed channel pressure drop. Membrane autopsy confirmed biofilm presence prior to the pressure drop increase, highlighting the advantage of early detection in implementing corrective measures. Timely intervention reduces operational costs and energy consumption in membrane-based processes.

Ginsenoside Rh2 Alleviate Sepsis-related Encephalopathy via Up-regulating Nrf2/HO-1 Pathway and Apoptosis Inhibition.

Sepsis patients are highly prone to sepsis-associated encephalopathy (SAE) complications, resulting in a high mortality rate. Recently, there has been no specific treatment for long-term improvement of cerebral function. Ginsenoside Rh2 is a form of steroidal saponins isolated from plant ginseng and has been shown to possess anti-inflammatory as well as neuroprotective characteristics; yet, the effect of ginsenoside Rh2 on SAE treatment is obscure. Accordingly, we proposed to investigate the effect of ginsenoside Rh2 in alleviating SAE damage. We established and utilized the SAE mouse model to determine the effect of Rh2 treatment on alleviating SAE. We determined the expression levels of Heme oxygenase-1(HO-1) and Nuclear factor erythroid 2-related factor 2 (Nrf2) as well as measured neural apoptosis by flow cytometry. Also, we quantified the levels of caspase-3, malondialdehyde (MDA), GSH-Px superoxide dismutase (SOD) and evaluated the animals' neural reflex function. First, used Rh2 to treat microglia BV2 and mouse neuron MN-c whether LPS exist or not, and then measured expression level of Iba-1, apoptotic rate, and ROS content applying flow cytometry. Also, we quantified the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). In comparison with the Sham group, the SAE model exhibited an elevated MDA content, caspase-3 activity, and cell apoptosis. On the other hand, the GSH-Px activity and SOD level were decreased along with a decreased neural reflex score. Our investigation concluded that Rh2 treatment significantly alleviated SAE damage and inhibited LPS-induced response via up-regulation of the Nrf2/HO-1 pathway to promote anti-oxidative stress capacity and inhibit neural cell apoptosis.

Development of the PD9-9 Monoclonal Antibody for Identifying Porcine Bone Marrow-Derived Dendritic Cells.

The purpose of this study was to develop a monoclonal antibody (mAb) that can identify porcine dendritic cells (DCs) that have differentiated from bone marrow progenitor cells. Hybridoma technology was used to obtain mAbs, and bone marrow-derived DCs (BMDCs) were employed as immunogens for producing antibodies. The generated PD9-9 mAbs exhibited considerable reactivity towards porcine BMDCs with applications in flow cytometry and immunostaining. The antibody was composed of heavy immunoglobulin gamma-1 chains and light kappa chains. The PD9-9 mAb recognized fully differentiated porcine BMDCs and cells undergoing DC differentiation. In contrast, bone marrow cells and macrophages were not recognized by PD9-9. In addition, the PD9-9 mAb promoted porcine DC proliferation. Consequently, the PD9-9 mAb may be a biomarker for porcine DCs and will be advantageous for investigating porcine DC biology.

Functional characterization of uncertain significance variants (VUS) in patients with familial hypercholesterolemia (FH) through the application of flow cytometry

Functional characterization of uncertain significance variants (VUS) in patients with familial hypercholesterolemia (FH) through the application of flow cytometry

Flow cytometric genome size estimation of Hungarian grass varieties

In Keszthely, the breeding of grass species have several decades long history. Among other things, the aim of the maintenance work of the registered varieties is to preserve the ploidy levels that have been reported in the variety descriptions. Flow cytometry is a fast, modern tool for examining plant DNA content. In our pilot study, we compared the genome size of the four grass species maintained at the MATE Georgikon Campus with the data of known samples of similar ploidy levels published for the given grass species in the international database. Our results showed a high degree of agreement with the theoretically expected values, supporting the applicability of flow cytometry in plant breeding and in variety maintenance.

Abstract A023: Stratification of bladder cancer patients with the use of flow cytometry is increasing the diagnostic potential of molecular classifiers

Abstract Recent research suggests the potential categorization of bladder cancer (BLCa) patients into molecular subtypes to aid in the selection of appropriate treatment strategies, be it conventional, targeted, or a combination thereof. However, the practical implementation of molecular classifiers has faced obstacles due to the intricate nature of gene regulation, tumor heterogeneity, and challenges related to tumor sampling caused by substantial stromal infiltration and tumor multifocality. Hence, there is a pressing need for more straightforward marker-based methodologies to assist in genomic and transcriptomic subtyping efforts. Our study addressed this gap by establishing and validating a panel of antibodies for application in flow cytometry (FCM) to enhance the stratification of BLCa patients. We curated a set of 17 FCM markers capable of delineating four cell clusters (epithelial, immune, endothelial, and stromal) and defining tumor classification (basal and luminal), immune infiltration, and hot-target expression on BLCa tissue samples. This FCM panel was deployed on ex vivo tissue samples treated with chemotherapy and targeted therapy agents. Initially tested on a cohort of 10 BLCa tissue samples, our validation of the FCM panel revealed correlations between marker expression and clinical-pathological features, such as the presence of metastasis and heightened immune and stromal infiltration. Notably, samples exhibiting lower claudin-4 levels displayed increased immune infiltration, while those with metastases at diagnosis showcased elevated basal marker expression. The translational potential of the FCM panel was demonstrated in ex vivo experiments where tissue slices from six patients were subjected to chemotherapy and targeted therapy agents, leading to observable shifts in marker expression post-treatment, particularly pronounced with chemotherapy. Noteworthy effects included reductions in epithelial cells expressing FGFR3 and EGFR following targeted therapy with erdafitinib or lapatinib, alongside alterations in the basal/luminal cell ratio indicated by increased CD44, CD90, and CD239 marker expressions. Our findings underscore the value of FCM in categorizing BLCa patients based on specific marker expression, supplementing conventional histological analyses to enhance cancer diagnosis, guide personalized therapeutic approaches, and monitor treatment efficacy. Citation Format: Martina Radic, Federico La Manna, Martina Minoli, Panagiotis Chouvardas, George N. Thalmann, Roland Seiler-Blarer, Marianna Kruithof-de Julio, Bernhard Kiss. Stratification of bladder cancer patients with the use of flow cytometry is increasing the diagnostic potential of molecular classifiers [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A023.

Stable, fluorescent markers for tracking synthetic communities and assembly dynamics

BackgroundAfter two decades of extensive microbiome research, the current forefront of scientific exploration involves moving beyond description and classification to uncovering the intricate mechanisms underlying the coalescence of microbial communities. Deciphering microbiome assembly has been technically challenging due to their vast microbial diversity but establishing a synthetic community (SynCom) serves as a key strategy in unravelling this process. Achieving absolute quantification is crucial for establishing causality in assembly dynamics. However, existing approaches are primarily designed to differentiate a specific group of microorganisms within a particular SynCom.ResultsTo address this issue, we have developed the differential fluorescent marking (DFM) strategy, employing three distinguishable fluorescent proteins in single and double combinations. Building on the mini-Tn7 transposon, DFM capitalises on enhanced stability and broad applicability across diverse Proteobacteria species. The various DFM constructions are built using the pTn7-SCOUT plasmid family, enabling modular assembly, and facilitating the interchangeability of expression and antibiotic cassettes in a single reaction. DFM has no detrimental effects on fitness or community assembly dynamics, and through the application of flow cytometry, we successfully differentiated, quantified, and tracked a diverse six-member SynCom under various complex conditions like root rhizosphere showing a different colonisation assembly dynamic between pea and barley roots.ConclusionsDFM represents a powerful resource that eliminates dependence on sequencing and/or culturing, thereby opening new avenues for studying microbiome assembly.CZiF6AaEp7jbjKaYRmvD15Video

A lasso and random forest model using flow cytometry data identifies primary myelofibrosis.

Thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis (PMF), prefibrotic/early (pre-PMF), and overt fibrotic PMF (overt PMF) are classical Philadelphia-Negative (Ph-negative) myeloproliferative neoplasms (MPNs). Differentiating between these types based on morphology and molecular markers is challenging. This study aims to clarify the application of flow cytometry in the diagnosis and differential diagnosis of classical MPNs. This study retrospectively analyzed the immunophenotypes, clinical characteristics, and laboratory findings of 211 Ph-negative MPN patients, including ET, PV, pre-PMF, overt PMF, and 47 controls. Compared to ET and PV, PMF differed in white blood cells, hemoglobin, blast cells in the peripheral blood, abnormal karyotype, and WT1 gene expression. PMF also differed from controls in CD34+ cells, granulocyte phenotype, monocyte phenotype, percentage of plasma cells, and dendritic cells. Notably, the PMF group had a significantly lower plasma cell percentage compared with other groups. A lasso and random forest model select five variables (CD34+CD19+cells and CD34+CD38- cells on CD34+cells, CD13dim+CD11b- cells in granulocytes, CD38str+CD19+/-plasma, and CD123+HLA-DR-basophils), which identify PMF with a sensitivity and specificity of 90%. Simultaneously, a classification and regression tree model was constructed using the percentage of CD34+CD38- on CD34+ cells and platelet counts to distinguish between ET and pre-PMF, with accuracies of 94.3% and 83.9%, respectively. Flow immunophenotyping aids in diagnosing PMF and differentiating between ET and PV. It also helps distinguish pre-PMF from ET and guides treatment decisions.

Chinese expert consensus on the application of flow cytometry for the detectin of circulating plasma cells in plasma cell disorders (2024)

Flow cytometry plays an important role in the diagnosis and treatment of plasma cell diseases, particularly in the detection of circulating plasma cells (CPCs) in the peripheral blood. A consensus about the normalized use of flow cytometry in detection of CPCs in peripheral blood in clinical practice has been achieved. This consensus is founded on evidence-based principles, which elucidates the timing and value of flow cytometry for the detection of CPCs in the monoclonal gammopathy of undetermined significance, smoldering myeloma, multiple myeloma, and plasma cell leukemia and standardizes flow cytometry in the detection of CPCs in plasma cell diseases.

GNAI3 mediated by Lin28A regulates lipopolysaccharide-induced inflammation and osteogenic differentiation in periodontal stem cells by mediating the NF-κB/NLRP3 inflammasome pathway

ObjectivesThe aim of this study was to investigate the regulatory role of G protein subunit alpha i3 (GNAI3) in periodontitis. DesignFollowing the induction of human periodontal ligament stem cells (hPDLSCs) with lipopolysaccharide (LPS), the mRNA and protein expressions of GNAI3 and Lin28A were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. The transfection efficiency of Oe-GNAI3 and sh-Lin28A was examined by virtue of RT-qPCR and western blot. With the application of ELISA and flow cytometry, the releases of inflammatory cytokines and cell apoptosis were appraised. Alkaline phosphatase (ALP) staining and alizarin red S (ARS) staining were conducted to evaluate osteogenic differentiation. Next, the binding ability of Lin28A with GNAI3 mRNA was estimated by radioimmunoprecipitation (RIP) assay while the stability of GNAI3 mRNA was assessed utilizing RT-qPCR. Western blot was employed for the measurement of inflammation-, apoptosis- and nuclear factor-kappaB (NF-κB)/NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome pathway-related proteins and osteogenic markers. ResultsThe expression of GNAI3 was down-regulated in LPS-induced hPDLSCs. After the transfection with Oe-GNAI3, the inflammation and apoptosis in LPS-induced hPDLSCs were inhibited while osteogenic differentiation was promoted. Moreover, Lin28A could stabilize GNAI3 mRNA and Lin28A knockdown significantly reduced GNAI3 expression. Further experiments verified that the inhibitory effects of GNAI3 overexpression on LPS-induced cellular inflammation and cell apoptosis as well as the promotive effects on osteogenic differentiation in hPDLSCs were all partially counteracted by Lin28A depletion, which may possibly be mediated via the regulation of the NF-κB/NLRP3 inflammasome pathway. ConclusionGNAI3 that mediated by Lin28A regulates the inflammation and osteogenic differentiation in LPS-induced hPDLSCs by mediating the NF-κB/NLRP3 inflammasome pathway.

Water‐Soluble Fluorescent Polymer Dyes with Tunable Emission Spectra for Flow Cytometry Applications

AbstractThe application of spectrally unique, bright, and water‐soluble fluorescent dyes is indispensable for the analysis of biological systems. Multiparameter flow cytometry is a powerful tool for characterization of mixed cell populations. To discriminate the different cell populations, they are typically stained by a set of fluorescent reagents, e.g., antibody‐fluorophore conjugates. The number of parameters which can be studied simultaneously strongly depends on the availability of reagents which can be differentiated by their spectral properties. In this study a series of fluorescent polymer dyes was developed, that can be excited with a single violet laser (405 nm) but distinguished by their unique emission spectra. The polyfluorene‐based polymers can be used on their own, or in combination with covalently bound small‐molecule dyes to generate energy transfer constructs to red‐shift the emission wavelength based on Förster resonance energy transfer (FRET). The polymer dyes were utilized in a biological flow cytometry assay by conjugating several of them to antibodies, demonstrating their effectiveness as reagents. This report represents the first systematic investigation of structure–property relationships for this type of fluorescent dyes.

Water-Soluble Fluorescent Polymer Dyes with Tunable Emission Spectra for Flow Cytometry Applications.

The application of spectrally unique, bright, and water-soluble fluorescent dyes is indispensable for the analysis of biological systems. Multiparameter flow cytometry is a powerful tool for characterization of mixed cell populations. To discriminate the different cell populations, they are typically stained by a set of fluorescent reagents, e.g., antibody-fluorophore conjugates. The number of parameters which can be studied simultaneously strongly depends on the availability of reagents which can be differentiated by their spectral properties. In this study a series of fluorescent polymer dyes was developed, that can be excited with a single violet laser (405 nm) but distinguished by their unique emission spectra. The polyfluorene-based polymers can be used on their own, or in combination with covalently bound small-molecule dyes to generate energy transfer constructs to red-shift the emission wavelength based on Förster resonance energy transfer (FRET). The polymer dyes were utilized in a biological flow cytometry assay by conjugating several of them to antibodies, demonstrating their effectiveness as reagents. This report represents the first systematic investigation of structure-property relationships for this type of fluorescent dyes.

Application of Flow Cytometry for Viability Assay of Mutants for Translation Termination Factors in the Yeast Saccharomyces cerevisiae

Application of Flow Cytometry for Viability Assay of Mutants for Translation Termination Factors in the Yeast Saccharomyces cerevisiae

Applications of Flow Cytometry in Drug Discovery and Translational Research.

Flow cytometry is a mainstay technique in cell biology research, where it is used for phenotypic analysis of mixed cell populations. Quantitative approaches have unlocked a deeper value of flow cytometry in drug discovery research. As the number of drug modalities and druggable mechanisms increases, there is an increasing drive to identify meaningful biomarkers, evaluate the relationship between pharmacokinetics and pharmacodynamics (PK/PD), and translate these insights into the evaluation of patients enrolled in early clinical trials. In this review, we discuss emerging roles for flow cytometry in the translational setting that supports the transition and evaluation of novel compounds in the clinic.

Examining the Effects of Asiaticoside on Dental Pulp Stem Cell Viability and Proliferation: A Promising Approach to Root Canal Treatment.

This study aims to evaluate the impact of asiaticoside (AC) on the viability and proliferation of dental pulp stem cells (DPSCs), considering the known negative effects of routinely used intracanal medicaments. This evaluation will be compared with the outcomes from using traditional intracanal medicaments, specifically triple antibiotic paste (TAP) and calcium hydroxide [Ca(OH)2]. The DPSCs were obtained from the third molars of an adult donor. The application of flow cytometry was employed to do a phenotypic analysis on DPSCs using CD90, CD73, CD105, CD34, CD14, and CD45 antibodies. The methylthiazol tetrazolium (MTT) assay was employed to assess cellular viability. The cells were treated with different concentrations of TAP and Ca(OH)2 (5, 2.5, 1, 0.5, and 0.25 mg/mL), along with AC (100, 50, 25, 12.5, and 6.25 µM). A cell proliferation rate was performed at 3, 5, and 7 days. The characterization of DPSCs was conducted by flow cytometry analysis, which verified the presence of mesenchymal cell surface antigen molecules (CD105, CD73, and CD90) and demonstrated the absence of hematopoietic markers (CD34, CD45, and CD14). Cells treated with concentrations over 0.5 mg/mL of TAP and Ca(OH)2 showed a notable reduction in cell viability in comparison to the untreated cells (p < 0.05). Additionally, the cells treated with different concentrations of AC 12.5, 6.25, 25, and 50 µM did not differ significantly from the untreated cells (p > 0.05). Nevertheless, cells treated with concentrations of 100 µM showed a significant reduction in viability compared to the untreated cells (p < 0.05). After a period of 7 days, it was noted that cells exposed to three different concentrations of AC (50, 25, and 12.5 µM) had a notable rise in cell density in comparison to TAP and Ca(OH)2 (p < 0.05). Furthermore, cells that were exposed to a concentration of 12.5 µM exhibited the highest cell density. The cellular viability of the AC-treated cells was superior to that of the TAP and Ca(OH)2-treated cells. Moreover, the AC with a concentration of 12.5 µM had the highest degree of proliferation. This study underscores the importance of evaluating alternative root canal medicaments and their effects on DPSCs' growth and vitality. The findings on AC, particularly its influence on the survival and proliferation of DPSCs, offer valuable insights for its probable use as an intracanal medication. This research contributes to the ongoing efforts to identify safer and more effective intracanal treatments, which are crucial for enhancing patient outcomes in endodontic procedures. How to cite this article: Alazemi MJ, Badawi MF, Elbeltagy MG, et al. Examining the Effects of Asiaticoside on Dental Pulp Stem Cell Viability and Proliferation: A Promising Approach to Root Canal Treatment. J Contemp Dent Pract 2024;25(2):118-127.

Dynamic establishment of recipient resident memory T cell repertoire after human intestinal transplantation

Understanding formation of the human tissue resident memory T cell (TRM) repertoire requires longitudinal access to human non-lymphoid tissues. By applying flow cytometry and next generation sequencing to serial blood, lymphoid tissue, and gut samples from 16 intestinal transplantation (ITx) patients, we assessed the origin, distribution, and specificity of human TRMs at phenotypic and clonal levels. Donor age ≥1 year and blood T cell macrochimerism (peak level ≥4%) were associated with delayed establishment of stable recipient TRM repertoires in the transplanted ileum. T cell receptor (TCR) overlap between paired gut and blood repertoires from ITx patients was significantly greater than that in healthy controls, demonstrating increased gut-blood crosstalk after ITx. Crosstalk with the circulating pool remained high for years of follow-up. TCR sequences identifiable in pre-Tx recipient gut but not those in lymphoid tissues alone were more likely to populate post-Tx ileal allografts. Clones detected in both pre-Tx gut and lymphoid tissue had distinct transcriptional profiles from those identifiable in only one tissue. Recipient T cells were distributed widely throughout the gut, including allograft and native colon, which had substantial repertoire overlap. Both alloreactive and microbe-reactive recipient T cells persisted in transplanted ileum, contributing to the TRM repertoire. Our studies reveal human intestinal TRM repertoire establishment from the circulation, preferentially involving lymphoid tissue counterparts of recipient intestinal T cell clones, including TRMs. We have described the temporal and spatial dynamics of this active crosstalk between the circulating pool and the intestinal TRM pool. This study was funded by the National Institute of Allergy and Infectious Diseases (NIAID) P01 grant AI106697.