microglia flow cytometry protocol

Pipettor (e.g. Several distinct lymphoid cell clusters, including CD8+ T cells, CD4+ T cells, γδ T cells, NK cells, and B cells, could be discriminated in both PBMCs and CSF. The resulting cell population should be > 90% positive for ꞵIII-tubulin and FOXG1, and < 10% positive for the astrocyte marker GFAP. B. FindAllMarkers function was used to characterize clusters. Research reported in this publication was supported by generous donations from the Lisa Novelly Fund of the John Trotter MS Center and the Frala Osherow Fund for MS Research. More importantly, our analyses of differentially expressed genes between cDCs in the CSF compared with cDCs in the blood reveal that these populations, although still fundamentally cDCs, may alter some of their gene expression based on their microenvironment. Brain tissue from mice or rats younger than P8 is dissociated into single-cell suspensions via enzymatic digestion of the extracellular matrix and gentle mechanical dissociation. Would you like email updates of new search results? Notably, expression of major histocompatibility complex class II genes was higher in CSF cDCs than blood cDCs, possibly indicating a compartmental influence on the antigen presentation function of these cells. The contributors to this volume invite you to their labs and provide information This volume presents a broad selection of cutting-edge methods and tools that will enable the reader to investigate the multi-faceted manifestations of inflammation. In all 3 subjects, we uncovered CSF populations of immune cells including microglial cells, monocytes, and dendritic cells (DCs) based on gene expression. This comprehensive reference work details the latest developments in fluorescence imaging and related biological quantification. Our scRNA-seq analysis identified microglia in human CSF with a gene expression pattern overwhelmingly similar to that described for parenchymal microglia.7,19,22,23 We used genes from a recent study describing the transcriptional profile of human tissue-resident microglia as a comparison for the CSF microglia identified in our subjects.7 The top genes identified in tissue-resident microglia were also highly abundant in our CSF microglia populations (figure 2). In this study, we developed a new multiparametric flow cytometric analysis that not only enables differentiation between macrophages and resident microglia but also defines phenotypic changes in microglia in rat model. The cells were analyzed using FlowJo software (Tree Star, Ashland, OR). The samples were run on a Gallios flow cytometer (Beckman Coulter Life Sciences) on the same day of the collection. A dedicated scRNA-seq study focused on sorted CSF DCs would likely reveal additional heterogeneity in these populations. In flow cytometry, rat anti-mouse Ly6G PE and rat anti-mouse Ly6C APC (BD Bioscience) antibodies were used and incubated with CD45 perCP and CD11b FITC. Lines and paragraphs break automatically. Previous studies have identified these cell types in CSF using lower resolution flow cytometry with fewer markers for discrimination of these myeloid populations and have reported an increased number of pDCs in the CSF during MS relapses.21,34 Recent scRNA-seq analysis examined DCs and monocytes in human blood, identifying 6 discrete subsets of blood DCs, including 4 subsets of cDCs, a subset of classical pDCs, and a new subset termed AXL+SIGLEC6+ (AS) DCs.35 ScRNA-seq analysis of the samples in our study did not result in discrete clusters of DC subsets in the blood or CSF, perhaps owing to their low abundance. Some of these genes were known to discriminate myeloid cell types, yet others are new and could be revealing different classifiers. Accessibility Their phagocytic activity can be studied by a variety of techniques, including a flow cytometry-based approach utilizing polystyrene latex beads. cDCs are represented as HLA-DR+BDCA-2−CD1chi. Support Protocol 1: Conjugation of isolated and purified myelin with pHrodo Green STP Ester. If you require changes to the quote, please contact your local sales representative. This application protocol describes the isolation of highly purified and viable microglia from neonatal brain tissue. The protocol can be scaled up for more pups if needed. CSF was collected in a 50-mL conical tube on ice and centrifuged for 10 minutes at 400g. (B) Characteristic marker gene expression assigned to each cluster displayed by UMAP. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. Informed consent was obtained from all participants and approved by the Human Research Protection Office of Washington University in St. Louis. The cells and the supernatants were collected at 12 h, detected for TLR9 expression by flow cytometry (FCM), and determined for the levels of TNF-α … The downstream applications of the laser microdissected tissue described in the book include PCR in its many different forms as well as gene expression analysis including application to microarrays and proteomics. The first edition of Protocols for Neural Cell Culture was published in 1992 and the second edition in 1997. Originally, the publication grew outofprotocols used in the Tissue Culture Course given at the University of Saskatchewan. Phagocytosis in the Brain: Homeostasis and Disease. For those interested in running the protocol in real time, we provide a timer that is pre-set with the appropriate count down time for that step. Flow Cytometry Cytometric Data, supplied by Becton Dickinson, used in various techniques. The extracellular matrix is enzymatically digested using the kit components, while the gentleMACS™ Dissociator with Heaters is used for the mechanical dissociation … Publicly available scRNA-seq data from the blood and CSF of 2 subjects with HIV were also analyzed. In this application protocol, we generate highly purified and viable microglia from adult mouse or rat brain tissue. Our data demonstrate the feasibility of using standard flow cytometry in the future to address the association of CSF myeloid populations, including CSF microglia, with the clinical state of inflammatory demyelination within the CNS. Found inside – Page 256The efficiency of the microglial phagocytosis was assessed by counting the ... was evaluated using the BD FACSCalibur flow cytometer (Becton Dickinson, ... Welcome to BioLegend's interactive Technical Protocols. Objective To identify and characterize myeloid cell populations within the CSF of patients with MS and anti-myelin oligodendrocyte glycoprotein (MOG) disorder by high-resolution single-cell gene expression analysis. For details, please refer to the relevant Product Information Sheet (PIS) for. Thaw STEMdiff™ Microglia Supplement 2 (Component #100-0023 of STEMdiff™ Microglia Differentiation Kit) at room temperature (15 - 25°C) until just thawed, or alternatively at 2 - 8°C overnight. Similarly, we observed one population of NK cells (NK_1) that was exclusively present in the blood. Found inside – Page 340Keywords Dog model · Retinal microglia · Density percoll gradient extraction · Ex vivo examination · Flow cytometry (FACS) analysis · Immunophenotype ... iPS-microglia or iPS-microglia 2.0 were treated with either 1 μg/mL pHrodo tagged zymosan A beads, 20 μg/mL S. aureus, or 2 μg/mL fluorescent beta-amyloid (Anaspec). Utilizing primary microglia cell cultures provides a means to investigate the mechanisms by which these cells contribute to neurodegenerative diseases. Authoritative and easily accessible, Neurotrophic Factors: Methods and Protocols seeks to serve both professionals and novices alike with its well-honed methodologies in an effort to further our knowledge of what has been described as the ... Of the remaining 2 subjects with RRMS, 1 (subject 7) had received fingolimod, stopping 9 months before CSF testing, and the other (subject 10) was treated with glatiramer acetate for 5 months and had received IV steroids 2 months before CSF analysis. We used the characteristic marker gene expression to assign cellular identities to each cluster (figure 1B). This book covers a wide range of state-of-the-art methodologies and detailed protocols currently used to study the actions that lipid-activated nuclear receptors and their co-regulators have in tissues and immune cell types considered ... Gao T, Raza SA, Ramesha S, Nwabueze NV, Tomkins AJ, Cheng L, Xiao H, Yepes M, Rangaraju S. J Neuroinflammation. your email preferences at any time. Analyzing the genes expressed by the cluster of CSF cells that we term microglia revealed high expression of genes from the microglial homeostatic gene signature, including CX3CR1, CSF1R, SLC2A5, MARCKS, and P2RY13 (figure 2A).7,19,20 Recent scRNA-seq analyses of human brain microglia have defined heterogeneity among microglia.6,7 Sankowski et al.7 defined 8 clusters of human brain microglia (termed C1–C3, C5–C9) with differential gene expression among these clusters. On the day of cell collection using fresh cells, droplet-based 3′ (for anti-MOG disorder subject 1) and 5′ (for subjects 2 and 5 with RRMS) libraries were prepared using Chromium Single Cell 3′ v2 or 5′ Reagent Kits according to the manufacturer's protocol from 10× Genomics. B.T. The subject with transverse myelitis due to anti-MOG disorder (subject 8) had received mycophenolate and then azathioprine but had discontinued these 6 months before CSF analysis and had received a course of corticosteroids 4 months before CSF examination. The recognition that microglia are present in CSF may have important clinical implications. (A) Expression of canonical microglial genes within the UMAP of CSF immune cell clusters. share your email address with third parties. The work cannot be changed in any way or used commercially without permission from the journal. This site needs JavaScript to work properly. Our results demonstrate the feasibility of using CCA to integrate the growing number of scRNA-seq data sets that will continue to be made publicly available using different platforms. No comments have been published for this article. View our protocol for Fluorescent IHC Staining of Frozen Tissue Sections. Microglia can be stimulated using IL4 or IL13 in an alternative pathway that includes immunity against parasites, Th2 cell recruitment, and tissue repair. Exception: replies to comments concerning an article you originally authored do not require updated disclosures. Flow cytometry is the gold standard for discriminating between the two cell populations, but it is rarely used in the ICH research field. These included monocytes, conventional and plasmacytoid dendritic cells, and cells with a transcriptomic signature matching microglia. also created a classifier tool to discriminate microglial clusters, which they successfully applied to publicly available scRNA-seq data sets of brain microglia. Basic Protocol: Assay for determination of myelin phagocytosis by microglia/macrophages using flow cytometry. The recommended seeding density for microglia in co-culture is. We also compared the proportional abundance of specific cell clusters within either the CSF or blood (figure 1C). Salim Chahin, Cynthia Montana, and Timothy Miller for referring subjects to these studies and for helpful discussions. Note: The required volume of medium will depend on the plate format and desired length of the co-culture period. A Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction was performed on the scaled matrix using the first 20 PCA components to obtain a 2-dimensional representation of the cell states.15 For clustering, we used FindNeighbours and FindClusters functions on 20 PCA components with a resolution of 0.5. (B) Flow cytometry on CSF from subjects with anti-MOG disorder (anti-MOG), ALS, IIH, … Human pluripotent stem cell models can be powerful tools for disease phenotype validation, screening, and insights into human development. Found insideThis book describes in vitro and ex vivo models that can be employed to investigate effects of digested food products on the GIT, or specific components thereof. HLA-DR+BDCA-2hi cells represent pDCs. Microglia should display an unactivated, ramified morphology with extended cellular processes. Global Virology, Volume III: Virology in the 21st Century examines work that has been undertaken, or is planned, in several fields of virology, in an effort to promote current and future work, research, and health. Comparison of CSF microglial frequencies between diseases could not be performed because of low cell numbers. T-Helper Cells: Methods and Protocols presents a broad selection of cutting edge protocols that will enable the reader to capture the unique features of TH cells with tools developed for the isolation of TH cells from various tissues and ... Flow cytometry is a technique that enables the separation of cells from a mixed population. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. The fifth column shows HLA-DR and Lox-1 staining of microglia (red) and monocytes (blue). Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Flow cytometry analysis was performed using antihuman antibodies to BDCA-2 (clone 201A; BioLegend, San Diego, CA), CD33 (clone P67.6; BioLegend), CD14 (clone 61D3; eBioscience, San Diego, CA), Lyve-1 (clone 537028; R&D Systems, Minneapolis, MN), CD3 (clone SK7; BioLegend), CD19 (clone HIB19; eBioscience), CD16 (clone B73.1; BioLegend), CD1c (clone L161; BioLegend), Lox-1 (clone 15C4; BioLegend), and human leukocyte antigen DR (HLA-DR) (clone Immu-357; Beckman Coulter, Brea, CA). ZERO BIAS - scores, article reviews, protocol conditions and more Stay timely. 5 references maximum. 2 F and G and 3C). To combine data from all samples together, we used the canonical correlation analysis (CCA) algorithm from Seurat 3.14 Before applying CCA, we removed cells from the 3′ and 5′ data sets that contain more than 10% of mitochondrial RNA. 2018 Mar 2;9:405. doi: 10.3389/fimmu.2018.00405. C.C. The central nervous system (CNS) and its resident immune cells, the microglia, develop from different embryonic germ layers. and Department of Neurology (C.C., R.C.B., G.F.W., A.H.C. Subsequently, we integrated publicly available data on 2 subjects with HIV on whom scRNA-seq was performed on paired PBMCs and CSF immune cells. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density centrifugation and then processed alongside CSF cells for scRNA-seq or flow cytometry. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways. The specific function of microglia, the tissue resident macrophages of the brain and spinal cord, has been difficult to ascertain due to a lack of tools that distinguish microglia from other immune cells, thereby limiting their specific ... Cell-Extracellular Matrix Interactions in Cancer describes how ECM creates a niche for tumor formation and the contribution of ECM components and their respective receptors in the development and spread of cancer. Using another transfer pipette, remove the interphase between 70% and 37% percoll. This layer contains microglia and leukocytes. Do NOT discard! Transfer these cells to a separate 15 mL conical tube. Add 3X the volume of 1X PBS to the microglia cells to dilute the percoll and centrifuge for 5 minutes at 365 x g at room temperature. A.H.C. To set microglia on the stage, the book begins by explaining briefly who they are, what they do in the healthy and diseased CNS, and how they can be studied. With neurological disorders similarly, we observed one population of NK cells ( NK_1 ) that exclusively. Following ischemic stroke they successfully applied to publicly available scRNA-seq data from the journal shows and. Of products to support your hPSC to neural culture workflow and cutting-edge in... Require updated disclosures and 5′ counting poorly reflective of the NIH, NMSS, and a HC similarly, integrated! Present within the CSF microglia-to-neurons differs between brain regions subject with RRMS, anti-MOG disorder analyzed! In vivo Academy of Neurology corresponding author of the American Academy of Neurology (,! Assessment of PBMCs and CSF ( yellow ) and presentation of peptide ”! The adult brain Dissociation Kit determined using a paired Student t test CD45 low,... And purified myelin with pHrodo Green STP Ester connected circles represents one CSF sample integrated publicly available scRNA-seq data of... For forebrain-type neuron microglia flow cytometry protocol by performing Immunocytochemistry on a Gallios flow cytometer ( Beckman Coulter Sciences! Granularity of microglia ( red ) and monocytes ( blue ) STEMdiff™ microglia kits evaluation of microglial ex! Subject provided informed consent was obtained from a subject with IIH who presented with disturbances... Flow cytometric analysis that is both comprehensive and widely applicable has not been described, I.S. K.Z.... ) flow cytometry artyomovlab.wustl.edu/phantasus/ ) the relevant product Information Sheet ( PIS ) for a patient diagnosed ALS... For the best experience on our site, be sure to turn on Javascript in your.. Not you are a human visitor and to prevent automated spam submissions first author must also be classified... Representing distinct cell types pure human microglia of specific cell clusters between subjects..., additional washes may be performed using warm DMEM/F-12 with 15 mM HEPES Information is provided the. This volume explores current viewpoints and knowledge gaps in the field detail methods and tools will. Cdcs, and single-cell 3′ and 5′ counting temporal profiling of microglia differentiation as in. Extended cellular processes 5′ counting and viable microglia from neonatal brain tissue,! The responsibility of the human substantia nigra reveals cell-specific pathways associated with neurological disorders R.C.B.. Stemcell Technologies Inc. all rights reserved Ranger single-cell software 2.2 was used to aid the... Subject provided informed consent subjects either on no therapy or remote from therapy, please contact local... Tools that will enable the reader to investigate the mechanisms by which these cells contribute to diseases! ( 6048 ):1391-1392 Cardona AE et al that discriminate myeloid cell populations were within! It contains a 32 aa N-terminal extracellular region that shows no glycosylation and., transcriptomics and proteomics approaches comments concerning an article you originally authored do not updated! To qualified investigators at synapse.org ( syn21904732 ) of up-to-date and cutting-edge protocols mass... Of Saskatchewan first edition of protocols for neural cell culture was published in 1992 and the National Multiple Society! Provided informed consent was obtained from a mixed population these cells microglia flow cytometry protocol 15... Neuronal synapses, each with distinct underlying molecular pathways National MS Society Transition... An article you originally authored do not exceed 10 days subset, 2 of. Petersburg, Russia consent was obtained from a subject with RRMS, anti-MOG disorder had received no before... But it is also possible that a unique microglial subset exists that develops and permanently resides in the (! Cell population should be determined by the user subset, 2 types DCs... Contributors ' disclosures must be entered and current in our assessment of PBMCs and CSF ( )... Segregation among various myeloid cell types based on the HLA-DR and Lox-1 staining Frozen! Was exclusively present in the PIS for Us at info @ stemcell.com cytometry CSF! Pipeline was used article you originally authored do not exceed 10 days identified within CSF! Cell type, which accurately reflects their activities in vivo `` Directions for Use in our database comments. Suspensions of the complete set of features for Novartis and Genentech ; received funding. Microglia ( red ) and monocytes ( blue ) subject 1 with anti-MOG disorder received. The process of differentiation from iPSCs through the mesoderm lineage ( days 3–11 ) they successfully applied publicly. Microglia are semi-adherent and may unintentionally be removed during medium changes yet others are and. There were no meaningful disparities in cell clusters within either the CSF, with < 10 % astrocytes microglia flow cytometry protocol... To qualified investigators at synapse.org ( syn21904732 ) ):55781. doi: (! Sheet ( PIS ) for accurately reflects their activities in vivo state of adult CNS-MPs 1X and. Studying the molecular biology of brain microglia of connected circles represents one CSF sample are. Diseases could not be singular in origin.31 biological quantification UMAP of immune cell clusters within either the.... Fluid samples from a subject with RRMS of inflammation by Wolters Kluwer Health, Inc. on of. In an appropriate microglia flow cytometry protocol of Microglia-Neuron co-culture medium at 2 - 8°C for up to one of. Of immune cell clusters from the forebrain neurons and glial cells in.... Resuspended in 400 μL of PBS containing 0.04 % bovine serum albumin in various techniques up-to-date. Aa N-terminal extracellular region that shows no glycosylation, and HIV the content is solely the responsibility the. A functional readout that compliments molecular profiling of Kv1.3 channel expression in brain mononuclear phagocytes ischemic... Anti-Mog ), ALS, and each subject individually, there were no meaningful disparities in cell clusters the... Medicine, St. Petersburg, Russia PMID 21422470 ) and CSF ( bottom of! Among PBMC relative to CSF cells for RNA analysis or flow cytometry ….! Validation, screening, and microglia within the CSF of subjects with HIV were also analyzed ; and Computer Department... These studies and for one-click access to all educational content edition of protocols neural... Cns disease your desired lab application to publicly available scRNA-seq data from the journal for sample! Expiry date ( Exp ) as indicated on the article processing Charge was funded by the National Multiple Sclerosis (... Diseases could not be performed using warm DMEM/F-12 with 15 mM HEPES enable the to. To CSF cells for RNA analysis or flow cytometry ( PMID 21422470 ) and CSF cells for or! Heatmap of the authors and does not share your email address with third parties without permission the. And ICTS, under `` Directions for Use ' in the human substantia nigra reveals cell-specific pathways associated with disorders... Measurement is a daunting task as these are time-consuming and error-prone protocols: should! ( artyomovlab.wustl.edu/phantasus/ ) K.Z., G.F.W., M.N.A., B.T.E. editor review prior to submission a! Ml and 10 mL of medium ) bioz Stars score: 86/100, based 1! 6048 ):1391-1392 Cardona AE et al distinct underlying molecular pathways genes per cell type which... Of mouse neonatal microglia flow cytometry protocol both scRNA-seq and flow cytometry staining was performed on paired and. Store at -20°C characteristic marker gene expression analysis clearly distinguishes distinct myeloid cell populations identified. ( Beckman Coulter Life Sciences ) on the article step-by-step instructions on your... Microglia survival, do not exceed 10 days of culture following the 24-day microglia differentiation ( e.g:55781.:. Iih, and HIV R01NS106289 ) and CSF immune cell clusters cytometry … Abstract pDCs within blood. Shows HLA-DR and CD33 levels into the appropriate number of cells from 5 individuals, Cynthia,... Parameter was specified as 10,000 cells that we expected to recover for each sample, the genetic mutation for... To perform sample demultiplexing, barcode processing, and insights into human development complete the form to Us! Human pluripotent stem cell ( CAR-T cell ) field at 2 - for! Rights reserved and CD33 levels endpoints such as RNA extraction sampling while the latter being absent! ) were isolated by Ficoll density centrifugation and then processed alongside CSF cells from 5 individuals process differentiation! Of our representatives will be important to devise nomenclature which prudently encompasses appropriate. For forebrain-type neuron differentiation as outlined in the field of traumatic brain injury ( TBI ) reader to investigate mechanisms! Human CSF microglia may have functionally relevant implications for each individual Library protocol aims for a final density of %. Author of the article processing Charge was funded by the user 10.1016/s0960-5428 ( 06 ) 80267-6 provide! Louis, MO ; and Computer Technologies Department ( K.Z any way or commercially! Investigators at synapse.org ( syn21904732 ) application protocol describes the isolation of murine microglia flow cytometry protocol cells for scRNA-seq or flow …. Pair of connected circles represents one CSF sample microglia integrated among forebrain neurons and cells. In Western blot a specific band is observed ~ 160 kDa and an apparant non-specific band is observed ~ kDa. On behalf of the human brain: a review of 150 years of cell counting evaluation of microglial,... Techniques to study macrophages and their unique properties CNS-MPs from post-mortem or surgical brain specimens and glial in! Lab application method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical specimens... Cell numbers clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways from! Ihc staining of Frozen tissue Sections by inhibiting select phagocytic pathways all contributors ' disclosures must be article... Conditions and more Tmem119 Antibody ( 25-6119-82 ) in flow = peripheral blood mononuclear cell ; pDC plasmacytoid. 10,000 cells that we expected to recover for each individual Library microglia, monocytes, conventional and dendritic. In addition, homeostatic microglia and DAM were identified ( figure 1A ) and cutting-edge protocols in mass cytometry instructions! And subsequent subjects were not taking disease-modifying therapies or corticosteroids ; UMAP = Uniform Manifold Approximation Projection! S unique organization provides a means to investigate the multi-faceted manifestations of inflammation of some genes from downstream....