RESUMEN

In food industry, Listeria monocytogenes contamination can occur accidentally despite the quality control of raw materials and factory. Decontamination processes or inhibitory effects of ingredients/additives in food products are set up to ensure compliance with hygiene and microbiological criteria. These actions represent stresses for the pathogenic agent, causing fluctuations in its physiological states. Moreover, during these environmental stresses, Listeria monocytogenes can enter in a viable but nonculturable (VBNC) state which is not detected by plate counting but by flow cytometry. This technique coupled with cell staining by fluorescent dyes offers the possibility to assess different physiological states based on different cellular parameters: enzymatic activity, transmembrane integrity, membrane potential, and respiratory activity. In this chapter, we present a method to assess the viability of foodborne pathogens using a double-staining principle based on the assessment of membrane integrity and intracellular esterase activity.

Asunto(s)

Citometría de Flujo , Listeria monocytogenes , Viabilidad Microbiana , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/fisiología , Citometría de Flujo/métodos , Microbiología de Alimentos/métodos , Colorantes Fluorescentes/química , Coloración y Etiquetado/métodos , Membrana Celular/metabolismo

RESUMEN

This chapter introduces protocols for culturing and maintaining Dictyostelium discoideum and methods for conducting virulence assays in this organism to study bacterial pathogenicity. It outlines advanced techniques, such as automated microscopy and flow cytometry, for detailed cellular analysis and traditional microbiological approaches. These comprehensive protocols will enable researchers to probe the virulence factors of pathogens like Klebsiella pneumoniae and to elucidate the details of host-pathogen interactions within a cost-effective and adaptable laboratory framework.

Asunto(s)

Dictyostelium , Citometría de Flujo , Klebsiella pneumoniae , Dictyostelium/microbiología , Citometría de Flujo/métodos , Klebsiella pneumoniae/patogenicidad , Fagocitosis , Virulencia , Interacciones Huésped-Patógeno , Microscopía/métodos

RESUMEN

Acute myeloid leukemia (AML) is a common type of acute leukemia (AL), belonging to malignant tumors of the hematopoietic system with the characteristics of rapid disease development, control with extreme difficulties, easy recurrence, poor prognosis, and incidence rate increasing with age. The traditionally diagnostic standard of French American British (FAB), being based on the morphological examination with high human subjectivity, can no longer meet the demand of clinical diagnosis and treatment of AML. Requirements of objective accuracy and low-dose sample, have become the indispensable method for AML diagnosis and monitoring prognosis. Flow cytometry is a modern technology that can quickly and accurately detect the series, antigen distribution, differentiation stage of AML cells, minimal residual lesions after AML therapy, so as to provide the great significance in guiding clinical diagnosis, hierarchical treatment, and prognosis judgement. This article will systematically elaborate on the application of flow cytometry in the diagnosis and classification of AML, and the detection of minimal residual lesions, thereby providing reference significance for dynamic monitoring and prognostic observation of AML with different immune subtypes of FAB.

Asunto(s)

Citometría de Flujo , Leucemia Mieloide Aguda , Neoplasia Residual , Humanos , Leucemia Mieloide Aguda/diagnóstico , Neoplasia Residual/diagnóstico

RESUMEN

Significance: Advancements in label-free microscopy could provide real-time, non-invasive imaging with unique sources of contrast and automated standardized analysis to characterize heterogeneous and dynamic biological processes. These tools would overcome challenges with widely used methods that are destructive (e.g., histology, flow cytometry) or lack cellular resolution (e.g., plate-based assays, whole animal bioluminescence imaging). Aim: This perspective aims to (1) justify the need for label-free microscopy to track heterogeneous cellular functions over time and space within unperturbed systems and (2) recommend improvements regarding instrumentation, image analysis, and image interpretation to address these needs. Approach: Three key research areas (cancer research, autoimmune disease, and tissue and cell engineering) are considered to support the need for label-free microscopy to characterize heterogeneity and dynamics within biological systems. Based on the strengths (e.g., multiple sources of molecular contrast, non-invasive monitoring) and weaknesses (e.g., imaging depth, image interpretation) of several label-free microscopy modalities, improvements for future imaging systems are recommended. Conclusion: Improvements in instrumentation including strategies that increase resolution and imaging speed, standardization and centralization of image analysis tools, and robust data validation and interpretation will expand the applications of label-free microscopy to study heterogeneous and dynamic biological systems.

Asunto(s)

Técnicas Histológicas , Microscopía , Animales , Citometría de Flujo , Procesamiento de Imagen Asistido por Computador

RESUMEN

Mass cytometry is a cutting-edge high-dimensional technology for profiling marker expression at the single-cell level, advancing clinical research in immune monitoring. Nevertheless, the vast data generated by cytometry by time-of-flight (CyTOF) poses a significant analytical challenge. To address this, we describe ImmCellTyper (https://github.com/JingAnyaSun/ImmCellTyper), a novel toolkit for CyTOF data analysis. This framework incorporates BinaryClust, an in-house developed semi-supervised clustering tool that automatically identifies main cell types. BinaryClust outperforms existing clustering tools in accuracy and speed, as shown in benchmarks with two datasets of approximately 4 million cells, matching the precision of manual gating by human experts. Furthermore, ImmCellTyper offers various visualisation and analytical tools, spanning from quality control to differential analysis, tailored to users' specific needs for a comprehensive CyTOF data analysis solution. The workflow includes five key steps: (1) batch effect evaluation and correction, (2) data quality control and pre-processing, (3) main cell lineage characterisation and quantification, (4) in-depth investigation of specific cell types; and (5) differential analysis of cell abundance and functional marker expression across study groups. Overall, ImmCellTyper combines expert biological knowledge in a semi-supervised approach to accurately deconvolute well-defined main cell lineages, while maintaining the potential of unsupervised methods to discover novel cell subsets, thus facilitating high-dimensional immune profiling.

Asunto(s)

Análisis de Datos , Citometría de Flujo , Análisis de la Célula Individual , Humanos , Citometría de Flujo/métodos , Análisis de la Célula Individual/métodos , Programas Informáticos , Análisis por Conglomerados

RESUMEN

PROBLEM: Endometrial immune cells are essential for maintaining homeostasis and the endometrial receptivity to embryo implantation. Understanding regional variations in endometrial immune cell populations is crucial for comprehending normal endometrial function and the pathophysiology of endometrial disorders. Despite previous studies focusing on the overall immune cell composition and function in the endometrium, regional variations in premenopausal women remain unclear. METHOD OF STUDY: Endometrial biopsies were obtained from four regions (anterior, posterior, left lateral, and right lateral) of premenopausal women undergoing hysteroscopy with no abnormalities. A 15-color human endometrial immune cell-focused flow cytometry panel was used for analysis. High-dimensional flow cytometry combined with a clustering algorithm was employed to unravel the complexity of endometrial immune cells. Additionally, multiplex immunofluorescent was performed for further validation. RESULTS: Our findings revealed no significant variation in the distribution and abundance of immune cells across different regions under normal conditions during the proliferative phase. Each region harbored similar immune cell subtypes, indicating a consistent immune microenvironment. However, when comparing normal regions to areas with focal hemorrhage, significant differences were observed. An increase in CD8+ T cells highlights the impact of localized abnormalities on the immune microenvironment. CONCLUSIONS: Our study demonstrates that the endometrial immune cell landscape is consistent across different anatomical regions during the proliferative phase in premenopausal women. This finding has important implications for understanding normal endometrial function and the pathophysiology of endometrial disorders.

Asunto(s)

Microambiente Celular , Endometrio , Humanos , Femenino , Endometrio/inmunología , Endometrio/patología , Adulto , Microambiente Celular/inmunología , Citometría de Flujo , Premenopausia/inmunología , Linfocitos T CD8-positivos/inmunología , Biopsia

RESUMEN

Introduction: Pediatric patients with unexplained bone marrow failure (BMF) are often categorized as aplastic anemia (AA). Based on the accepted hypothesis of an auto-immune mechanism underlying AA, immune suppressive therapy (IST) might be effective. However, due to the lack of diagnostic tools to identify immune AA and prognostic markers to predict IST response together with the unequaled curative potential of hematopoietic stem cell transplantation (HSCT), most pediatric severe AA patients are momentarily treated by HSCT if available. Although several studies indicate oligoclonal T-cells with cytotoxic activities towards the hematopoietic stem cells, increasing evidence points towards defective inhibitory mechanisms failing to inhibit auto-reactive T-cells. Methods: We aimed to investigate the role of NK- and B-cells in seven pediatric AA patients through a comprehensive analysis of paired bone marrow and peripheral blood samples with spectral flow cytometry in comparison to healthy age-matched bone marrow donors. Results: We observed a reduced absolute number of NK-cells in peripheral blood of AA patients with a skewed distribution towards CD56bright NK-cells in a subgroup of patients. The enriched CD56bright NK-cells had a lower expression of CD45RA and TIGIT and a higher expression of CD16, compared to healthy donors. Functional analysis revealed no differences in degranulation. However, IFN-γ production and perforin expression of NK-cells were reduced in the CD56bright-enriched patient group. The diminished NK-cell function in this subgroup might underly the auto-immunity. Importantly, NK-function of AA patients with reduced CD56bright NK-cells was comparable to healthy donors. Also, B-cell counts were lower in AA patients. Subset analysis revealed a trend towards reduction of transitional B-cells in both absolute and relative numbers compared to healthy controls. As these cells were previously hypothesized as regulatory cells in AA, decreased numbers might be involved in defective inhibition of auto-reactive T-cells. Interestingly, even in patients with normal distribution of precursor B-cells, the transitional compartment was reduced, indicating partial differentiation failure from immature to transitional B-cells or a selective loss. Discussion: Our findings provide a base for future studies to unravel the role of transitional B-cells and CD56bright NK-cells in larger cohorts of pediatric AA patients as diagnostic markers for immune AA and targets for therapeutic interventions.

Asunto(s)

Anemia Aplásica , Linfocitos B , Inmunofenotipificación , Células Asesinas Naturales , Humanos , Células Asesinas Naturales/inmunología , Anemia Aplásica/inmunología , Anemia Aplásica/terapia , Niño , Masculino , Femenino , Linfocitos B/inmunología , Adolescente , Preescolar , Citometría de Flujo

RESUMEN

Introduction: Immunogenicity refers to the ability of a substance, such as a therapeutic drug, to elicit an immune response. While beneficial in vaccine development, undesirable immunogenicity can compromise the safety and efficacy of therapeutic proteins by inducing anti-drug antibodies (ADAs). These ADAs can reduce drug bioavailability and alter pharmacokinetics, necessitating comprehensive immunogenicity risk assessments starting at early stages of drug development. Given the complexity of immunogenicity, an integrated approach is essential, as no single assay can universally recapitulate the immune response leading to the formation of anti-drug antibodies. Methods: To better understand the Dendritic Cell (DC) contribution to immunogenicity, we developed two flow cytometry-based assays: the DC internalization assay and the DC activation assay. Monocyte-derived dendritic cells (moDCs) were generated from peripheral blood mononuclear cells (PBMCs) and differentiated over a five-day period. The internalization assay measured the accumulation rate of therapeutic antibodies within moDCs, while the activation assay assessed the expression of DC activation markers such as CD40, CD80, CD86, CD83, and DC-SIGN (CD209). To characterize these two assays further, we used a set of marketed therapeutic antibodies. Results: The study highlights that moDCs differentiated for 5 days from freshly isolated monocytes were more prone to respond to external stimuli. The internalization assay has been shown to be highly sensitive to the molecule tested, allowing the use of only 4 donors to detect small but significant differences. We also demonstrated that therapeutic antibodies were efficiently taken up by moDCs, with a strong correlation with their peptide presentation on MHC-II. On the other hand, by monitoring DC activation through a limited set of activation markers including CD40, CD83, and DC-SIGN, the DC activation assay has the potential to compare a series of compounds. These two assays provide a more comprehensive understanding of DC function in the context of immunogenicity, highlighting the importance of both internalization and activation processes in ADA development. Discussion: The DC internalization and activation assays described here address key gaps in existing immunogenicity assessment methods by providing specific and reliable measures of DC function. The assays enhance our ability to pre-clinically evaluate the immunogenic potential of biotherapeutics, thereby improving their safety and efficacy. Future work should focus on further validating these assays and integrating them into a holistic immunogenicity risk assessment framework.

Asunto(s)

Células Dendríticas , Células Dendríticas/inmunología , Humanos , Citometría de Flujo , Medición de Riesgo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Células Cultivadas , Diferenciación Celular/inmunología , Monocitos/inmunología

RESUMEN

Flow cytometry is a vital tool in biomedical research and laboratory medicine. However, its accuracy is often compromised by undesired fluctuations in fluorescence intensity. While fluorescence lifetime imaging microscopy (FLIM) bypasses this challenge as fluorescence lifetime remains unaffected by such fluctuations, the full integration of FLIM into flow cytometry has yet to be demonstrated due to speed limitations. Here we overcome the speed limitations in FLIM, thereby enabling high-throughput FLIM flow cytometry at a high rate of over 10,000 cells per second. This is made possible by using dual intensity-modulated continuous-wave beam arrays with complementary modulation frequency pairs for fluorophore excitation and acquiring fluorescence lifetime images of rapidly flowing cells. Moreover, our FLIM system distinguishes subpopulations in male rat glioma and captures dynamic changes in the cell nucleus induced by an anti-cancer drug. FLIM flow cytometry significantly enhances cellular analysis capabilities, providing detailed insights into cellular functions, interactions, and environments.

Asunto(s)

Citometría de Flujo , Glioma , Citometría de Flujo/métodos , Animales , Ratas , Glioma/diagnóstico por imagen , Glioma/patología , Glioma/metabolismo , Masculino , Microscopía Fluorescente/métodos , Línea Celular Tumoral , Imagen Óptica/métodos , Humanos , Núcleo Celular/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Colorantes Fluorescentes/química

RESUMEN

Cell-cell interactions are essential for the proper functioning of multicellular organisms. For example, T cells interact with antigen-presenting cells (APCs) through specific T-cell receptor (TCR)-antigen interactions during an immune response. Fluorescence-activated droplet sorting (FADS) is a high-throughput technique for efficiently screening cellular interaction events. Unfortunately, current droplet sorting instruments have significant limitations, most notably related to analytical throughput and complex operation. In contrast, commercial fluorescence-activated cell sorters offer superior speed, sensitivity, and multiplexing capabilities, although their use as droplet sorters is poorly defined and underutilized. Herein, we present a universally applicable and simple-to-implement workflow for generating double emulsions and performing multicolor cell sorting using a commercial FACS instrument. This workflow achieves a double emulsion detection rate exceeding 90%, enabling multicellular encapsulation and high-throughput immune cell activation sorting for the first time. We anticipate that the presented droplet sorting strategy will benefit cell biology laboratories by providing access to an advanced microfluidic toolbox with minimal effort and cost investment.

Asunto(s)

Emulsiones , Citometría de Flujo , Citometría de Flujo/métodos , Emulsiones/química , Humanos , Colorantes Fluorescentes/química , Linfocitos T/citología , Color , Separación Celular/métodos , Animales

RESUMEN

BACKGROUND: Aplastic anemia (AA) is an immune-mediated syndrome characterized by bone marrow failure. Therefore, comprehending the cellular profile and cell interactions in affected patients is crucial. METHODS: Human peripheral blood mononuclear cells (PBMCs) were collected from both healthy donors (HDs) and AA patients, and analyzed using multicolor flow cytometry. Utilizing the FlowSOM and t-SNE dimensionality reduction technique, we systematically explored and visualized the major immune cell alterations in AA. This analysis provided a foundation to further investigate the subtypes of cells exhibiting significant changes. RESULTS: Compared to HDs, peripheral blood from patients with AA exhibits a marked reduction in CD56Dim natural killer (NK) cells, which also show diminished functionality. Conversely, an increase in NK-like CD56+ monocytes, which possess compromised functionality. Along with a significant reduction in myeloid-derived suppressor cells (MDSCs), which show recovery post-treatment. Additionally, MDSCs serve as effective clinical markers for distinguishing between acquired aplastic anemia (AAA) and congenital aplastic anemia (CAA). Our comprehensive analysis of correlations among distinct immune cell types revealed significant associations between NKBri cells and CD8+ T cell subsets, as well as between NKDim cells and CD4+ T cells, these results highlight the intricate interactions and correlations within the immune cell network in AA. CONCLUSION: Our study systematically elucidates the pronounced immune dysregulation in patients with AA. The detailed mapping of the immune landscape not only provides crucial insights for basic research but also holds promise for enhancing the accuracy of diagnoses and the effectiveness of timely therapeutic interventions in clinical practice. Consequently, this could potentially reduce the high mortality rate associated with AA.

Asunto(s)

Anemia Aplásica , Células Asesinas Naturales , Humanos , Anemia Aplásica/inmunología , Células Asesinas Naturales/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Adulto , Femenino , Citometría de Flujo/métodos , Células Supresoras de Origen Mieloide/inmunología , Masculino , Persona de Mediana Edad , Adulto Joven , Anciano

RESUMEN

In scenarios where yeast and bacterial cells coexist, it is of interest to simultaneously quantify the concentrations of both cell types, since traditional methods used to determine these concentrations individually take more time and resources. Here, we compared different methods for quantifying the fuel ethanol Saccharomyces cerevisiae PE-2 yeast strain and cells from the probiotic Lactiplantibacillus plantarum strain in microbial suspensions. Individual suspensions were prepared, mixed in 1:1 or 100:1 yeast-to-bacteria ratios, covering the range typically encountered in sugarcane biorefineries, and analyzed using bright field microscopy, manual and automatic Spread-plate and Drop-plate counting, flow cytometry (at 1:1 and 100:1 ratios), and a Coulter Counter (at 1:1 and 100:1 ratios). We observed that for yeast cell counts in the mixture (1:1 and 100:1 ratios), flow cytometry, the Coulter Counter, and both Spread-plate options (manual and automatic CFU counting) yielded statistically similar results, while the Drop-plate and microscopy-based methods gave statistically different results. For bacterial cell quantification, the microscopy-based method, Drop-plate, and both Spread-plate plating options and flow cytometry (1:1 ratio) produced no significantly different results (p > .05). In contrast, the Coulter Counter (1:1 ratio) and flow cytometry (100:1 ratio) presented results statistically different (p < .05). Additionally, quantifying bacterial cells in a mixed suspension at a 100:1 ratio wasn't possible due to an overlap between yeast cell debris and bacterial cells. We conclude that each method has limitations, advantages, and disadvantages. ONE-SENTENCE SUMMARY: This study compares methods for simultaneously quantifying yeast and bacterial cells in a mixed sample, highlighting that in different cell proportions, some methods cannot quantify both cell types and present distinct advantages and limitations regarding time, cost, and precision.

Asunto(s)

Microbiología Industrial , Saccharomyces cerevisiae , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/citología , Microbiología Industrial/métodos , Citometría de Flujo/métodos , Recuento de Colonia Microbiana/métodos , Carga Bacteriana/métodos , Saccharum/microbiología , Microscopía/métodos

RESUMEN

Impaired clearance of amyloid ß (Aß) in late-onset Alzheimer's disease (AD) affects disease progression. The role of peripheral monocytes in Aß clearance from the central nervous system (CNS) is unclear. We use a flow cytometry assay to identify Aß-binding monocytes in blood, validated by confocal microscopy, Western blotting, and mass spectrometry. Flow cytometry immunophenotyping and correlation with AD biomarkers are studied in 150 participants from the AIBL study. We also examine monocytes in human cerebrospinal fluid (CSF) and their migration in an APP/PS1 mouse model. The assay reveals macrophage-like Aß-binding monocytes with high phagocytic potential in both the periphery and CNS. We find lower surface Aß levels in mild cognitive impairment (MCI) and AD-dementia patients compared to cognitively unimpaired individuals. Monocyte infiltration from blood to CSF and migration from CNS to peripheral lymph nodes and blood are observed. Here we show that Aß-binding monocytes may play a role in CNS Aß clearance, suggesting their potential as a biomarker for AD diagnosis and monitoring.

Asunto(s)

Enfermedad de Alzheimer , Péptidos beta-Amiloides , Disfunción Cognitiva , Progresión de la Enfermedad , Ratones Transgénicos , Monocitos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/sangre , Humanos , Monocitos/metabolismo , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/líquido cefalorraquídeo , Animales , Femenino , Anciano , Masculino , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/líquido cefalorraquídeo , Ratones , Anciano de 80 o más Años , Biomarcadores/líquido cefalorraquídeo , Biomarcadores/sangre , Biomarcadores/metabolismo , Citometría de Flujo , Modelos Animales de Enfermedad , Fagocitosis , Persona de Mediana Edad

RESUMEN

Malassezia is a lipophilic commensal yeast that resides mainly on the mammalian skin and is also found to associate with the internal organs. Dysbiosis of Malassezia is related to several diseases and often escapes detection as it is difficult to culture and maintain. Malassezia cell wall differs from other budding yeasts like S. cerevisiae due to the difference in the lipid content and is difficult to transform. In this study, we present a methodology to stain Malassezia's nucleus and perform cell cycle studies. However, staining presents a challenge due to its exceptionally thick cell wall with high lipid content, hindering conventional methods. Our novel methodology addresses this challenge and enables the staining of the Malassezia nucleus with a low background. This would allow researchers to visualize the overall nuclear health specifically nuclear morphology and analyze DNA content, crucial for cell cycle progression. By employing DNA-specific dyes like DAPI or Hoechst, we can observe the nuclear structure, and using PI we can differentiate cells in distinct cell cycle phases using techniques like flow cytometry. This novel staining methodology unlocks the door for in-depth cell cycle analysis in Malassezia which has challenged us through ages being refractory to genetic manipulations, paving the way for a deeper understanding of this commensal fungus and its potential role in human health.

Asunto(s)

Ciclo Celular , Núcleo Celular , Malassezia , Coloración y Etiquetado , Núcleo Celular/metabolismo , Humanos , Coloración y Etiquetado/métodos , Citometría de Flujo/métodos , Pared Celular/metabolismo

RESUMEN

The study of microorganism interactions is important for understanding the organization and functioning of microbial consortia. Additionally, the interaction between yeast and bacteria is of interest in the field of health and nutrition area for the development of probiotics. To investigate these microbial interactions at the cellular and molecular levels, a simple, reliable, and quantitative method is proposed. We demonstrated that flow cytometry enables the measurement of interactions at a single-cell level by detecting and counting yeast cells with bound fluorescent bacteria. Imaging flow cytometry revealed that the number of bacteria attached to yeast followed a Gaussian distribution whose maximum reached 14 bacterial cells using a clinical Escherichia coli strain E22 and the laboratory yeast strain BY4741. We found that the dynamics of adhesion resemble a Langmuir adsorption model, albeit it is a rapid and almost irreversible process. This adhesion is dependent on the mannose-specific type 1 fimbriae, as E. coli mutants lacking these appendages no longer adhere to yeast. However, this type 1 fimbriae-dependent adhesion could involve additional yeast cell wall factors, since the interaction between bacteria and yeast mutants with altered mannan content remained comparable to that of wild-type yeast. In summary, flow cytometry is an appropriate method for studying bacteria-yeast adhesion, as well as for the high-throughput screening of candidate molecules likely to promote or counteract this interaction.

Asunto(s)

Adhesión Bacteriana , Escherichia coli , Citometría de Flujo , Saccharomyces cerevisiae , Citometría de Flujo/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo

RESUMEN

Heteroplasmic mitochondrial DNA (mtDNA) variants accumulate as humans age, particularly in the stem-cell compartments, and are an important contributor to age-related disease. Mitochondrial dysfunction has been observed in osteoporosis and somatic mtDNA pathogenic variants have been observed in animal models of osteoporosis. However, this has never been assessed in the relevant human tissue. Mesenchymal stem cells (MSCs) are the progenitors to many cells of the musculoskeletal system and are critical to skeletal tissues and bone vitality. Investigating mtDNA in MSCs could provide novel insights into the role of mitochondrial dysfunction in osteoporosis. To determine if this is possible, we investigated the landscape of somatic mtDNA variation in MSCs through a combination of fluorescence-activated cell sorting and single-cell next-generation sequencing. Our data show that somatic heteroplasmic variants are present in individual patient-derived MSCs, can reach high heteroplasmic fractions and have the potential to be pathogenic. The identification of somatic heteroplasmic variants in MSCs of patients highlights the potential for mitochondrial dysfunction to contribute to the pathogenesis of osteoporosis.

Asunto(s)

ADN Mitocondrial , Células Madre Mesenquimatosas , Humanos , Células Madre Mesenquimatosas/metabolismo , ADN Mitocondrial/genética , Osteoporosis/genética , Osteoporosis/patología , Osteoporosis/metabolismo , Mitocondrias/metabolismo , Mitocondrias/genética , Análisis de la Célula Individual , Secuenciación de Nucleótidos de Alto Rendimiento , Femenino , Heteroplasmia/genética , Masculino , Citometría de Flujo , Variación Genética , Persona de Mediana Edad

RESUMEN

Protoplast sorting and purification methods are powerful tools enabling the enrichment of cellular subpopulations for basic and applied studies in plant sciences. Fluorescence-activated protoplast sorting (FAPS) is an efficient method to isolate specific protoplast populations based on innate features (size and autofluorescence) or expression of fluorescent proteins. FAPS-based methods have recently been deployed in single-cell purification for single-cell RNA sequencing-based transcriptional profiling studies. Protoplast sorting methods integrated with the ability to culture and recover whole plants add value to functional genomics and gene editing applications. Enriching cells expressing nucleases linked to fluorescent proteins can maximize knockout or knockin editing efficiencies and minimize toxic and off-target effects. Here, we report the protocol for protoplast preparation, sterile cell sorting, culture, and downstream regeneration of plants from canola protoplasts. This protocol can be successfully applied to all totipotent protoplast methods that can regenerate into whole plants. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Preparation of transfected canola protoplasts for sorting Basic Protocol 2: Fluorescence-activated protoplast sorting Basic Protocol 3: Bead culture of sorted protoplasts and recovery of plantlets.

Asunto(s)

Brassica napus , Citometría de Flujo , Protoplastos , Regeneración , Protoplastos/metabolismo , Brassica napus/genética , Brassica napus/citología , Brassica napus/metabolismo , Citometría de Flujo/métodos

RESUMEN

The side population (SP) cells are identified through Hoechst 33342 staining and analyzed using flow cytometry (FCM). The Hoechst SP method is utilized for the isolation of stem cells based on the dye efflux properties of ATP-binding cassette (ABC) transporters. The method was initially employed for the identification and isolation of hematopoietic stem cells (HSCs), but it has now evolved to primarily focus on the identification and isolation of cancer stem cells (CSCs). The traditional detection method of FCM uses a 355 nm laser to excite the dye to detect SP cells. Through this study, we have successfully identified alternative approaches for dye excitation that can effectively replace the detection of SP cells using a 355 nm laser. This is achieved through the utilization of high-power 375 nm or 405 nm lasers. This allows us to exercise enhanced selectivity in the detection of SP cells rather than being solely limited to the 355 nm laser flow cytometry.

Asunto(s)

Bencimidazoles , Citometría de Flujo , Células de Población Lateral , Citometría de Flujo/métodos , Bencimidazoles/química , Células de Población Lateral/citología , Células de Población Lateral/metabolismo , Humanos , Colorantes Fluorescentes/química , Animales , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/metabolismo

RESUMEN

Human PBMC-based assays are often used as biomarkers for the diagnosis and prognosis of disease, as well as for the prediction and tracking of response to biological therapeutics. However, the development and use of PBMC-based biomarker assays is often limited by poor reproducibility. Complex immunological assays can be further complicated by variation in cell handling before analysis, especially when using cryopreserved cells. Variation in postthaw viability is further increased if PBMC isolation and cryopreservation are done more than a few hours after collection. There is currently a lack of evidence-based standards for the minimal PBMC viability or "fitness" required to ensure the integrity and reproducibility of immune cell-based assays. In this study, we use an "induced fail" approach to examine the effect of thawed human PBMC fitness on four flow cytometry-based assays. We found that cell permeability-based viability stains at the time of thawing did not accurately quantify cell fitness, whereas a combined measurement of metabolic activity and early apoptosis markers did. Investigation of the impact of different types and levels of damage on PBMC-based assays revealed that only when cells were >60-70% live and apoptosis negative did biomarker values cease to be determined by cell fitness rather than the inherent biology of the cells. These data show that, to reproducibly measure immunological biomarkers using cryopreserved PBMCs, minimal acceptable standards for cell fitness should be incorporated into the assay protocol.

Asunto(s)

Supervivencia Celular , Criopreservación , Citometría de Flujo , Leucocitos Mononucleares , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Citometría de Flujo/métodos , Citometría de Flujo/normas , Reproducibilidad de los Resultados , Biomarcadores/metabolismo , Apoptosis

RESUMEN

Background: Many research laboratories have long-term repositories of cryopreserved peripheral blood mononuclear cells (PBMC), which are costly to maintain but are of uncertain utility for immunological studies after decades in storage. This study investigated preservation of cell surface phenotypes and in-vitro functional capacity of PBMC from viraemic HIV+ patients and healthy seronegative control subjects, after more than 20 years of cryopreservation. Methods: PBMC were assessed by 18-colour flow cytometry for major lymphocyte subsets within T, B, NK, and dendritic cells and monocytes. Markers of T-cell differentiation and activation were compared with original immunophenotyping performed in 1995/1996 on fresh blood at the time of collection. Functionality of PBMC was assessed by culture with influenza antigen or polyclonal T-cell activation, to measure upregulation of activation-induced CD25 and CD134 (OX40) on CD4 T cells and cytokine production at day 2, and proliferative CD25+ CD4 blasts at day 7. RNA was extracted from cultures containing proliferating CD4+ blast cells, and intracellular HIV RNA was measured using short amplicons for both the Double R and pol region pi code assays, whereas long 4-kbp amplicons were sequenced. Results: All major lymphocyte and T-cell subpopulations were conserved after long-term cryostorage, except for decreased proportions of activated CD38+HLA-DR+ CD4 and CD8 T cells in PBMC from HIV+ patients. Otherwise, differences in T-cell subpopulations between recent and long-term cryopreserved PBMC primarily reflected donor age-associated or HIV infection-associated effects on phenotypes. Proportions of naïve, memory, and effector subsets of T cells from thawed PBMC correlated with results from the original flow cytometric analysis of respective fresh blood samples. Antigen-specific and polyclonal T-cell responses were readily detected in cryopreserved PBMC from HIV+ patients and healthy control donors. Intracellular HIV RNA quantitation by pi code assay correlated with original plasma viral RNA load results. Full-length intracellular and supernatant-derived amplicons were generated from 5/12 donors, and sequences were ≥80% wild-type, consistent with replication competence. Conclusions: This unique study provides strong rationale and validity for using well-maintained biorepositories to support immunovirological research even decades after collection.

Asunto(s)

Criopreservación , Infecciones por VIH , Inmunofenotipificación , Leucocitos Mononucleares , ARN Viral , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Leucocitos Mononucleares/metabolismo , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Activación de Linfocitos/inmunología , Masculino , Adulto , Femenino , Citometría de Flujo