Comprehensive phenotyping of endothelial cells using flow cytometry 2: Human.
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ABSTRACT:
SUBMITTER: Grant D
PROVIDER: S-EPMC8628257 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Impedance flow cytometry (IFC) is a versatile lab-on-chip technology which enables fast and label-free analysis of pollen grains in various plant species, promising new research possibilities in agriculture and plant breeding. Hazelnut is a monoecious, anemophilous species, exhibiting sporophytic self-incompatibility. Its pollen is dispersed by wind in midwinter when temperatures are still low and relative humidity is usually high. Previous research found that hazelnut can be characterized by high degrees of pollen sterility following a reciprocal chromosome translocation occurring in some cultivated genotypes. In this study, IFC was used for the first time to characterize hazelnut pollen biology. IFC was validated via dye exclusion in microscopy and employed to (i) follow pollen hydration over time to define the best pre-hydration treatment for pollen viability evaluation; (ii) test hazelnut pollen viability and sterility on 33 cultivars grown in a collection field located in central Italy, and two wild hazelnuts. The accessions were also characterized by their amount and distribution of catkins in the tree canopy. Pollen sterility rate greatly varied among hazelnut accessions, with one main group of highly sterile cultivars and a second group, comprising wild genotypes and the remaining cultivars, producing good quality pollen. The results support the hypothesis of recurring reciprocal translocation events in Corylus avellana cultivars, leading to the observed gametic semi-sterility. The measured hazelnut pollen viability was also strongly influenced by pollen hydration (R adj2 = 0.83, P ≤ 0.0001) and reached its maximum at around 6 h of pre-hydration in humid chambers. Viable and dead pollen were best discriminated at around the same time of pollen pre-hydration, suggesting that high humidity levels are required for hazelnut pollen to maintain its functionality. Altogether, our results detail the value of impedance flow cytometry for high throughput phenotyping of hazelnut pollen. Further research is required to clarify the causes of pollen sterility in hazelnut, to confirm the role of reciprocal chromosome translocations and to investigate its effects on plant productivity.
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Project description:The reconstitution of basic cellular functions in micrometer-sized liposomes has led to a surge of interest in the construction of synthetic cells. Microscopy and flow cytometry are powerful tools for characterizing biological processes in liposomes with fluorescence readouts. However, applying each method separately leads to a compromise between information-rich imaging by microscopy and statistical population analysis by flow cytometry. To address this shortcoming, we here introduce imaging flow cytometry (IFC) for high-throughput, microscopy-based screening of gene-expressing liposomes in laminar flow. We developed a comprehensive pipeline and analysis toolset based on a commercial IFC instrument and software. About 60 thousands of liposome events were collected per run starting from one microliter of the stock liposome solution. Robust population statistics from individual liposome images was performed based on fluorescence and morphological parameters. This allowed us to quantify complex phenotypes covering a wide range of liposomal states that are relevant for building a synthetic cell. The general applicability, current workflow limitations, and future prospects of IFC in synthetic cell research are finally discussed.
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Project description:T regulatory cells (Tregs) are a cell subset that can suppress immune responses to maintain homeostasis and self-tolerance. In some scenarios, the immunosuppressive nature could be associated to other pathological developments such as autoimmune diseases and cancers. Due to the importance of Tregs in disease pathogenesis, we developed and validated an 11-color flow cytometry panel for phenotypic and functional detection of Treg markers using healthy human donor peripheral blood mononuclear cells (PBMCs). Our panel contains 4 Treg surface proteins and 2 functional cytokines as well as T-lymphocyte lineage markers CD3, CD4, and CD8. Our data shows an increase in expression of markers CD25, FoxP3, CTLA4, GITR and intracellular cytokines IL4 and TGF? when comparing unstimulated samples to CD3/CD28 bead stimulated samples. This 11-color panel can be used to functionally evaluate immunosuppressive Tregs in human PBMC samples.
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Project description:There is an increasing interest for monitoring circulating myeloid-derived suppressor cells (MDSCs) in cancer patients, but there are also divergences in their phenotypic definition. To overcome this obstacle, the Cancer Immunoguiding Program under the umbrella of the Association of Cancer Immunotherapy is coordinating a proficiency panel program that aims at harmonizing MDSC phenotyping. After a consultation period, a two-stage approach was designed to harmonize MDSC phenotype. In the first step, an international consortium of 23 laboratories immunophenotyped 10 putative MDSC subsets on pretested, peripheral blood mononuclear cells of healthy donors to assess the level of concordance and define robust marker combinations for the identification of circulating MDSCs. At this stage, no mandatory requirements to standardize reagents or protocols were introduced. Data analysis revealed a small intra-laboratory, but very high inter-laboratory variance for all MDSC subsets, especially for the granulocytic subsets. In particular, the use of a dead-cell marker altered significantly the reported percentage of granulocytic MDSCs, confirming that these cells are especially sensitive to cryopreservation and/or thawing. Importantly, the gating strategy was heterogeneous and associated with high inter-center variance. Overall, our results document the high variability in MDSC phenotyping in the multicenter setting if no harmonization/standardization measures are applied. Although the observed variability depended on a number of identified parameters, the main parameter associated with variation was the gating strategy. Based on these findings, we propose further efforts to harmonize marker combinations and gating parameters to identify strategies for a robust enumeration of MDSC subsets.
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Project description:Human peripheral blood mononuclear cells (PBMCs) originate from hematopoietic stem cells in the bone marrow, which mainly includes lymphocytes (T cells, B cells, and natural killer cells) and monocytes. Cryopreserved PBMCs providing biobank resources are crucial for clinical application or scientific research. Here, we used flow cytometry to explore the influence of long-term cryopreservation on the quality of PBMCs with the aim of providing important evidence for the effective utilization of biobank resources. The PBMCs were isolated from the peripheral blood, which was collected from volunteers in the hospital. After long-term cryopreservation in liquid nitrogen, we analyzed the changes in cell numbers, viability, and multiple subtypes of PBMCs and studied the apoptosis, proliferation, activation, function, and status of T cells in comparison with freshly isolated PBMCs by flow cytometry, and then further tracked the effects of long-term cryopreservation on the same sample. Although the different cell types in the PBMCs dynamically changed compared with those in the freshly isolated samples, PBMC recovery and viability remained stable after long-term cryopreservation, and the number of most innate immune cells (e.g., monocytes and B cells) was significantly reduced compared to that of the freshly isolated PBMCs or long-term cryopreserved PBMCs; more importantly, the proportion of T cell subtypes, apoptosis, proliferation, and functional T cells, except for Tregs, were not affected by long-term cryopreservation. However, the proportions of activated T, naïve T, central memory T, effector T, and effector memory T cells dynamically changed after long-term cryopreservation. This article provides important evidence for the effective utilization of biobank resources. Long-term cryopreserved PBMCs can be partly used as biological resources for clinical research or basic studies, but the effect of cryopreservation on PBMCs should be considered when selecting cell samples, especially in research relating to activating or inhibiting function.
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Project description:IntroductionThe information content of multiparametric flow cytometry experiments is routinely underexploited given the paucity of adequate tools for unbiased comprehensive data analysis that can be applied successfully and independently by immunologists without computational training.MethodsWe aimed to develop a tool that allows straightforward access to the entire information content of any given flow cytometry panel for immunologists without special computational expertise. We used a data analysis approach which accounts for all mathematically possible combinations of markers in a given panel, coded the algorithm and applied the method to mined and self-generated data sets.ResultsWe developed Flow Plex, a straightforward computational tool that allows unrestricted access to the information content of a given flow cytometry panel, enables classification of human samples according to distinct immune phenotypes, such as different forms of autoimmune uveitis, acute myeloid leukemia vs "healthy", "old" vs "young", and facilitates the identification of cell populations with potential biologic relevance to states of disease and health.ConclusionsWe provide a tool that allows immunologists and other flow cytometry users with limited bioinformatics skills to extract comprehensive, unbiased information from flow cytometry data sets.
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Project description:Endothelial cells in the peripheral circulation are rare events that require technically rigorous approaches for detection by flow cytometry. Visualization of these cells has been even more demanding, as this has historically required extensive enrichment and processing prior to attempting imaging. As a result, few, if any, examples exist on images of peripheral blood circulating endothelial cells (CECs) that include verification of the cell lineage both phenotypically and genomically. In this study, we have devised a method whereby CECs can be directly visualized after lysis of red blood cells and staining, without pre-enrichment or additional processing. Peripheral blood is stained with CD45, CD146, CD3, Hoechst, and DAPI to permit identification of CD146 positive, nonleukocyte, nucleated, and live cells that fit the description of CECs. These cells are imaged using the Amnis ImageStream(X), an imaging flow cytometer. Genomic verification of the endothelial nature of these cells is accomplished by using an aliquot of the same stained samples for sorting CECs using similar gating strategies. This proof of principle of direct imaging of CECs by imaging flow cytometry will permit studies to be conducted heretofore not possible, as the ImageStream(X) has the capability of detecting additional fluorochromes other than those used to identify the CECs. Such potential investigations include antigen colocalization or capping, autophagy and apoptosis, morphologic changes in response to therapy, and others. Thus, this method will enable a broad range of novel studies to be conducted using CECs as surrogates of the endothelium.
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Project description:Hematopoietic stem cells are the source of all inflammatory cell types. Discovery of specific cell surface markers unique to human hematopoietic stem (HSC) and progenitor (HSPC) cell populations has facilitated studies of their development from stem cells to mature cells. The specific marker profiles of HSCs and HSPCs can be used to understand their role in human inflammatory diseases. The goal of this study is to simultaneously measure HSCs and HSPCs in normal human venous blood using multicolor flow cytometry. Our secondary aim is to determine how G-CSF mobilization alters the quantity of each HSC and HSPC population. Here we show that cells within the CD34+ fraction of human venous blood contains cells with the same cell surface markers found in human bone marrow samples. Mobilization with G-CSF significantly increases the quantity of total CD34+ cells, blood borne HSCs, multipotent progenitors, common myeloid progenitors, and megakaryocyte erythroid progenitors as a percentage of total MNCs analyzed. The increase in blood borne common lymphoid and granulocyte macrophage progenitors with G-CSF treatment did not reach significance. G-CSF treatment predominantly increased the numbers of HSCs and multipotent progenitors in the total CD34+ cell population; common myeloid progenitors and megakaryocyte erythroid progenitors were enriched relative to total MNCs analyzed, but not relative to total CD34+ cells. Our findings illustrate the utility of multicolor flow cytometry to quantify circulating HSCs and HSPCs in venous blood samples from human subjects. © 2016 International Clinical Cytometry Society.
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Project description:Purified Retinal Ganglion Cells (RGCs) for in vitro study have been a valuable tool in the study of neural regeneration and in the development of therapies to treat glaucoma. Traditionally, RGCs have been isolated from early postnatal rats and mice, and more recently from human in vitro derived retinal organoids using a two-step immunopanning technique based upon the expression of Thy-1. This technique, however, limits the time periods from which RGCs can be isolated, missing the earliest born RGCs at which time the greatest stage of axon growth occurs, as well as being limited in its use with models of retinal degeneration as Thy-1 is downregulated following injury. While fluorescence associated cell sorting (FACS) in combination with new optogenetically labeled RGCs would be able to overcome this limitation, the use of traditional FACS sorters has been limited to genomic and proteomic studies, as RGCs have little to no survival post-sorting. Here we describe a new method for RGC isolation utilizing a combined immunopanning-fluorescence associated cell sorting (IP-FACS) protocol that initially depletes macrophages and photoreceptors, using immunopanning to enrich for RGCs before using low-pressure FACS to isolate these cells. We demonstrate that RGCs isolated via IP-FACS when compared to RGCs isolated via immunopanning at the same age have similar purity as measured by antibody staining and qRT-PCR; survival as measured by live dead staining; neurite outgrowth; and electrophysiological properties as measured by calcium release response to glutamate. Finally, we demonstrate the ability to isolate RGCs from early embryonic mice prior to the expression of Thy-1 using Brn3b-eGFP optogenetically labeled cells. This method provides a new approach for the isolation of RGCs for the study of early developed RGCs, the study of RGC subtypes and the isolation of RGCs for cell transplantation studies.
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Project description:A better understanding of the phenotypic heterogeneity of protoplasts requires a comprehensive analysis of the morphological and metabolic characteristics of many individual cells. In this study, we developed a microfluidic flow cytometry with fluorescence sensor for functional characterization and phenotyping of protoplasts to allow an unbiased assessment of the influence of environmental factors at the single cell level. First, based on the measurement of intracellular homeostasis of reactive oxygen species (ROS) with a DCFH-DA dye, the effects of various external stress factors such as H2O2, temperature, ultraviolet (UV) light, and cadmium ions on intracellular ROS accumulation in Arabidopsis mesophyll protoplasts were quantitatively investigated. Second, a faster and stronger oxidative burst was observed in Petunia protoplasts isolated from white petals than in those isolated from purple petals, demonstrating the photoprotective role of anthocyanins. Third, using mutants with different endogenous auxin, we demonstrated the beneficial effect of auxin during the process of primary cell wall regeneration. Moreover, UV-B irradiation has a similar accelerating effect by increasing the intracellular auxin level, as shown by double fluorescence channels. In summary, our work has revealed previously underappreciated phenotypic variability within a protoplast population and demonstrated the advantages of a microfluidic flow cytometry for assessing the in vivo dynamics of plant metabolic and physiological indices at the single-cell level.
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