Name: assessing retinal microglial phagocytic function in vivo using a flow cytometry-based assay
Uploaded: 2016-10-18T15:00:00
Description: Watch this Scientific Journal Video about Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay at JoVE.com

Salome Murinello is supported by American Diabetes Association grant #1-16-PDF-072. This work was supported by grants to Martin Friedlander from the National Institutes of Health (National Eye Institute EY11254 and EY22025) and the Lowy Medical Research Institute.

Alle dyr blev behandlet i overensstemmelse med de etiske retningslinjer fastsat af Scripps Research Institute. 1. Forberedelse af materialer til injektion <ol><li> Sterilisere en 33 G kanyle og sprøjte: demontere og autoklave ved 115 ο C. Forbered nåle til injektion af stigende i sterilt phosphatbufret saltvand (PBS). </li><li> Tø fluorescensmærkede partikler ved stuetemperatur i 5 - 10 min. Forbered partikel opløsning til injektion ved rekonstitution til en 50 mg / ml opløsning i sterilt PBS...

<ol>
	<li>Gomez-Nicola, D., Perry, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+dynamics+and+role+in+the+healthy+and+diseased+brain:+a+paradigm+of+functional+plasticity.">Microglial dynamics and role in the healthy and diseased brain: a paradigm of functional plasticity.</a> Neuroscientist. 21, 169-184 (2015).</li><li>Tremblay, M. E., Lowery, R. L., Majewska, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+interactions+with+synapses+are+modulated+by+visual+experience.">Microglial interactions with synapses are modulated by visual experience.</a> PLoS Biol. 8, e1000527 (2010).</li><li>Schafer, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+sculpt+postnatal+neural+circuits+in+an+activity+and+complement-dependent+manner.">Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner.</a> Neuron. 74, 691-705 (2012).</li><li>Sierra, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+shape+adult+hippocampal+neurogenesis+through+apoptosis-coupled+phagocytosis.">Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis.</a> Cell Stem Cell. 7, 483-495 (2010).</li><li>Sierra, A., Abiega, O., Shahraz, A., Neumann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Janus-faced+microglia:+beneficial+and+detrimental+consequences+of+microglial+phagocytosis.">Janus-faced microglia: beneficial and detrimental consequences of microglial phagocytosis.</a> Front Cell Neurosci. 7 (6), (2013).</li><li>Chan, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD33+modulates+TREM2:+convergence+of+Alzheimer+loci.">CD33 modulates TREM2: convergence of Alzheimer loci.</a> Nat Neurosci. 18, 1556-1558 (2015).</li><li>Murinello, S., Mullins, R. F., Lotery, A. J., Perry, V. H., Teeling, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fcgamma+receptor+upregulation+is+associated+with+immune+complex+inflammation+in+the+mouse+retina+and+early+age-related+macular+degeneration.">Fcgamma receptor upregulation is associated with immune complex inflammation in the mouse retina and early age-related macular degeneration.</a> Invest Ophthalmol Vis Sci. 55, 247-258 (2014).</li><li>Ransohoff, R. M., Perry, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+physiology:+unique+stimuli,+specialized+responses.">Microglial physiology: unique stimuli, specialized responses.</a> Annu Rev Immunol. 27, 119-145 (2009).</li><li>Derecki, N., Cronk, J., Kipnis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+phagocytic+function+in+primary+murine+microglia.">Assay of phagocytic function in primary murine microglia.</a> Protoc Exchange. , (2012).</li><li>Fricker, M., Oliva-Martin, M. J., Brown, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+phagocytosis+of+viable+neurons+by+microglia+activated+with+LPS+or+Abeta+is+dependent+on+calreticulin/LRP+phagocytic+signalling.">Primary phagocytosis of viable neurons by microglia activated with LPS or Abeta is dependent on calreticulin/LRP phagocytic signalling.</a> J Neuroinflammation. 9, 196 (2012).</li><li>Koenigsknecht-Talboo, J., Landreth, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+phagocytosis+induced+by+fibrillar+beta-amyloid+and+IgGs+are+differentially+regulated+by+proinflammatory+cytokines.">Microglial phagocytosis induced by fibrillar beta-amyloid and IgGs are differentially regulated by proinflammatory cytokines.</a> J Neurosci. 25, 8240-8249 (2005).</li><li>Neher, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+microglial+phagocytosis+is+sufficient+to+prevent+inflammatory+neuronal+death.">Inhibition of microglial phagocytosis is sufficient to prevent inflammatory neuronal death.</a> J Immunol. 186, 4973-4983 (2011).</li><li>Fricker, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MFG-E8+mediates+primary+phagocytosis+of+viable+neurons+during+neuroinflammation.">MFG-E8 mediates primary phagocytosis of viable neurons during neuroinflammation.</a> J Neurosci. 32, 2657-2666 (2012).</li><li>Mildner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+and+non-redundant+roles+of+microglia+and+myeloid+subsets+in+mouse+models+of+Alzheimer's+disease.">Distinct and non-redundant roles of microglia and myeloid subsets in mouse models of Alzheimer's disease.</a> J Neurosci. 31, 11159-11171 (2011).</li><li>Neher, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+executes+delayed+neuronal+death+after+focal+brain+ischemia.">Phagocytosis executes delayed neuronal death after focal brain ischemia.</a> Proc Natl Acad Sci U S A. 110, E4098-E4107 (2013).</li><li>Perego, C., Fumagalli, S., De Simoni, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+pattern+of+expression+and+colocalization+of+microglia/macrophage+phenotype+markers+following+brain+ischemic+injury+in+mice.">Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice.</a> J Neuroinflammation. 8, 174-174 (2011).</li><li>Preissler, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+microglial+phagocytosis+in+GPR34-deficient+mice.">Altered microglial phagocytosis in GPR34-deficient mice.</a> Glia. 63, 206-215 (2015).</li><li>Hughes, M. M., Field, R. H., Perry, V. H., Murray, C. L., Cunningham, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+in+the+degenerating+brain+are+capable+of+phagocytosis+of+beads+and+of+apoptotic+cells,+but+do+not+efficiently+remove+PrP(Sc),+even+upon+LPS+stimulation.">Microglia in the degenerating brain are capable of phagocytosis of beads and of apoptotic cells, but do not efficiently remove PrP(Sc), even upon LPS stimulation.</a> Glia. 58, 2017-2030 (2010).</li><li>Westenskow, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+flow+cytometry+to+compare+the+dynamics+of+photoreceptor+outer+segment+phagocytosis+in+iPS-derived+RPE+cells.">Using flow cytometry to compare the dynamics of photoreceptor outer segment phagocytosis in iPS-derived RPE cells.</a> Invest Ophthalmol Vis Sci. 53, 6282-6290 (2012).</li><li>London, A., Benhar, I., Schwartz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retina+as+a+window+to+the+brain-from+eye+research+to+CNS+disorders.">The retina as a window to the brain-from eye research to CNS disorders.</a> Nat Rev Neurol. 9, 44-53 (2013).</li><li>Fritzenwanger, M., Jung, C., Goebel, B., Lauten, A., Figulla, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+short-term+systemic+hypoxia+on+phagocytosis,+cytokine+production,+and+transcription+factor+activation+in+peripheral+blood+cells.">Impact of short-term systemic hypoxia on phagocytosis, cytokine production, and transcription factor activation in peripheral blood cells.</a> Mediators Inflamm. , 429501 (2011).</li><li>Ragsdale, R. L., Grasso, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+spectrofluorometric+assay+for+quantitating+yeast+phagocytosis+in+cultures+of+murine+peritoneal+macrophages.">An improved spectrofluorometric assay for quantitating yeast phagocytosis in cultures of murine peritoneal macrophages.</a> J Immunol Methods. 123, 259-267 (1989).</li><li>Stokes, L., Surprenant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+regulation+of+the+P2X4+receptor+in+alveolar+macrophages+by+phagocytosis+and+classical+activation.">Dynamic regulation of the P2X4 receptor in alveolar macrophages by phagocytosis and classical activation.</a> Eur J Immunol. 39, 986-995 (2009).</li><li>Kotani, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraoperative+modulation+of+alveolar+macrophage+function+during+isoflurane+and+propofol+anesthesia.">Intraoperative modulation of alveolar macrophage function during isoflurane and propofol anesthesia.</a> Anesthesiology. 89, 1125-1132 (1998).</li><li>Xu, X., Feng, J., Zuo, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+preconditioning+reduces+the+rat+NR8383+macrophage+injury+induced+by+lipopolysaccharide+and+interferon+gamma.">Isoflurane preconditioning reduces the rat NR8383 macrophage injury induced by lipopolysaccharide and interferon gamma.</a> Anesthesiology. 108, 643-650 (2008).</li><li>abd-el-Basset, E., Fedoroff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+bacterial+wall+lipopolysaccharide+(LPS)+on+morphology,+motility,+and+cytoskeletal+organization+of+microglia+in+cultures.">Effect of bacterial wall lipopolysaccharide (LPS) on morphology, motility, and cytoskeletal organization of microglia in cultures.</a> J Neurosci Res. 41, 222-237 (1995).</li><li>Menon, V., Thomas, R., Ghale, A. R., Reinhard, C., Pruszak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+protocols+for+surface+and+intracellular+antigen+analyses+of+neural+cell+types.">Flow cytometry protocols for surface and intracellular antigen analyses of neural cell types.</a> J Vis Exp. , (2014).</li><li>Jackson, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+vulnerability+to+neurodegenerative+disease:+the+curious+case+of+Prion+Protein.">Selective vulnerability to neurodegenerative disease: the curious case of Prion Protein.</a> Dis Model Mech. 7, 21-29 (2014).</li><li>O'Koren, E. G., Mathew, R., Saban, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+mapping+reveals+that+microglia+and+recruited+monocyte-derived+macrophages+are+definitively+distinguishable+by+phenotype+in+the+retina.">Fate mapping reveals that microglia and recruited monocyte-derived macrophages are definitively distinguishable by phenotype in the retina.</a> Sci Rep. 6, 20636 (2016).</li><li>Usui, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+and+Eye+Disease.">Angiogenesis and Eye Disease.</a> Annu Rev Vision. 1, 155-184 (2015).</li></ol>

Der er tre kritiske trin i denne fremgangsmåde: (1) intravitreal injektion af fluorescensmærkede partikler; (2) høst og klargøring af retinavæv; og (3) flowcytometrisk analyse. Vi anbefaler, at forskere praktiserer intravitreale injektioner før udøvelse af fremgangsmåden vi præsenterer her. Albinomus (fx BALB / c) og en farvet opløsning (f.eks fluorescensmærkede partikler) kan anvendes til nem visualisering af nålen og injiceret opløsning. Intravitreale injektioner er udfordrende, og hvis ...

Det overordnede mål med denne metode er at præcist at vurdere og kvantificere in vivo mikroglial fagocytose. Mikroglia er de væv stationære makrofager i centralnervesystemet (CNS). De udfører en række funktioner til at sikre opretholdelse af vævshomeostase. Disse omfatter immun overvågning, sekretion af neurotrofiske faktorer og af afgørende betydning, fagocytose 1. Mikroglial fagocytose er nøglen i flere vigtige begivenheder under udviklingen af hjernen og nethinden, såsom fagocytose af irrelevante synapser (synaptisk beskæring) og fjernelse af apoptotiske neuroner 2-4. Endvidere mikroglial fagocytose af beskadigede eller apoptotiske neuroner, cellerester og invaderende mikrober er blevet vist, at være afgørende for at opretholde CNS homeostase gennem voksenalderen 5. Endelig har mikroglial fagocytose blevet impliceret i patogenesen af ​​flere neurodegenerative sygdomme, herunder Alzheimers sygdom og aldersrelateretmakuladegeneration, hvor det er blevet foreslået, at defekt eller utilstrækkelig fagocytisk kapacitet kan bidrage til opbygning af amyloid-p (Ap) plaques og drusen henholdsvis 6,7. Mikroglial funktion er stramt reguleret af deres mikromiljø, navnlig ved opløselige faktorer, såsom tumor-vækstfaktor β eller celle-celle-interaktioner. Neuroner konstitutivt udtrykker flere celleoverfladeligander, såsom CD200 og CX3CL1, mens mikroglia udelukkende udtryk de respektive receptorer CD200R og CX3CR1. Disse receptorer indeholder immunoreceptorfusionsmolekyler tyrosin-baserede hæmning motiver (ITIM&#39;er) i deres intracellulære del. Disse inhibitor receptorer er kritisk for at forhindre over-stimulation af mikroglia, som kan bidrage til neuroinflammation. Således under normale fysiologiske betingelser, celle-celle interaktioner mellem neuroner og mikroglia holde mikroglia i en hvilende tilstand. Under vævsskade imidlertid neuroner kan nedregulere expression af disse ligander, fjerne deres inhiberende virkning på mikroglia aktivering. Microgliale funktion (herunder fagocytose) er således tæt knyttet til deres mikromiljø 8. Alligevel til dato, er der ingen standardiserede assays til at studere mikroglia fagocytose i en fysiologisk kontekst eller på en måde, der fuldt ud replikerer deres CNS mikromiljø. Adskillige assays er blevet udviklet til at måle fagocytisk aktivitet af mikroglia in vitro, hvor primære mikroglia eller microglia cellelinier dyrkes med målceller (f.eks apoptotiske neuroner) eller fluorescensmærkede perler. Target optagelse derefter vurderes ved hjælp af fluorescerende billeddannelse mikroskopi eller flowcytometri 9-12. Disse assays tillader afprøvning af hvordan farmakologisk eller genetisk manipulation kan påvirke mikroglial fagocytose og mens informative, ikke fuldt ud replikere komplekset in vivo miljø. Indirekte metoder til at undersøge mikroglial fagocytosein vivo er blevet rapporteret: disse er opnået ved farvning af molekyler menes at være involveret i fagocytose (fx CD68), vurdere fysisk nærhed af mikroglia og mål for fagocytose (f.eks kompromitteret neuroner eller synaptiske elementer), eller ved immunhistokemisk påvisning af fagocyterende mål inden mikrogliaceller (f.eks Ap) 13-17. To studier har brugt mere direkte metoder til at vurdere mikroglia fagocytose in vivo. Hughes og kolleger har brugt billeddannelsesteknikker til at måle mikroglial optagelse af perler leveret via det intrakranielle rute 18. Sierra et al. Udviklet en raffineret metode til kvantitative vurderinger mikroglia fagocytose af apoptotiske celler under anvendelse af komplekse billeddannelsesteknikker 4. Men disse metoder involverer komplicerede protokoller for vævspræparat, sektionering, billedbehandling og analyse. Vi har tidligere anvendt flowcytometrisk analyse for at vurdere fagocytose af fotoreceptor udER segmenter af retinale pigmenterede epithelium (RPE) celler i kultur 19. Her beskriver vi en protokol til hurtigt at vurdere optagelsen af fluorescens-mærkede partikler ved retinale mikroglia som et kvantitativt mål for in vivo microglia fagocytose. Protokollen vi beskriver her tillader pålidelig og kvantitativ måling af retinal mikroglial fagocytose i knap seks timer i tre kritiske trin: (1) intravitrealt levering af fluorescensmærkede partikler, (2) høst og klargøring af retinavæv, og (3) flow cytometrisk analyse. Den metode, vi har udviklet er en robust metode til vurdering mikroglial fagocytose i nethinden, og det med held kan anvendes til at teste, hvor forskellige forbindelser eller genetisk manipulation ændre denne nøgle mikroglial funktion i fysiologiske indstillinger. Som et specialiseret område i CNS, nethinden er en let tilgængelig modelsystem til at studere mikroglia funktion 20. Selv om denne fremgangsmåde blev udviklet i than øje, mener vi, det kan være nyttigt for alle neuroforskere efterforsker microglia fagocytotisk funktion.

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			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>Discontinued</td>
			<td></td>
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			<td>Hamilton&#160;syringe, 600 series</td>
			<td>Sigma</td>
			<td>26702</td>
			<td></td>
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			<td>33 gauge, Small Hub RN NDL, 0.5 in, point style 4 - 12o</td>
			<td>Hamilton</td>
			<td>7803-05</td>
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			<td>Zymosan A (S. cerevisiae) BioParticles, Alexa Fluor 488 conjugate</td>
			<td>ThermoFisher Scientific</td>
			<td>Z-23373</td>
			<td>Prepare immediately before injection</td>
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			<td>DPBS</td>
			<td>Corning</td>
			<td>21-030-CV</td>
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			<td>Dumont #5/45 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td>Need two</td>
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			<td>Dumont #5SF Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
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			<td>Vannas Spring Scissors - 3mm Cutting Edge</td>
			<td>Fine Science Tools</td>
			<td>15000-10</td>
			<td>Curved</td>
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			<td>Neural Tissue Dissociation Kit &#8211; Postnatal Neurons</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-802</td>
			<td></td>
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		<tr>
			<td>5 mL Polystyrene Round-bottom Tube</td>
			<td>Falcon</td>
			<td>352054</td>
			<td></td>
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			<td>96 well U-bottom plate</td>
			<td>Falcon</td>
			<td>353077</td>
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			<td>Stain Buffer (BSA)</td>
			<td>BD Biosciences</td>
			<td>554657</td>
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			<td>CD11b-BV650 Antibody</td>
			<td>BioLegend</td>
			<td>101259</td>
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			<td>Ly6C-APC-Cy7</td>
			<td>BioLegend</td>
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			<td>Ly6G-PE-Cy7</td>
			<td>BioLegend</td>
			<td>127617</td>
			<td></td>
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			<td>Propidium Iodide</td>
			<td>BD Biosciences</td>
			<td>556463</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified anti-mouse CD16/32 Antibody</td>
			<td>BioLegend</td>
			<td>101301</td>
			<td></td>
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assessing retinal microglial phagocytic function in vivo using a flow cytometry-based assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Her beskriver vi en metode til hurtigt og pålideligt kvantificere antallet af fagocytiske retinale mikroglia i en fysiologisk indstilling ved hjælp af flow cytometrisk analyse (figur 2). Denne metode kan tilpasses til at teste virkningen af forbindelser og / eller genetisk manipulation på den fagocytiske kapacitet af mikroglia (figur 3A, 3B). Den kan også anvendes i unge (10 - 20 dage postnatal) eller voksne mus (figur 3C). ...

Watch this Scientific Journal Video about Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay at JoVE.com

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Mikroglial fagocytose er kritisk for opretholdelse af vævshomeostase og utilstrækkelig fagocyterende funktion er blevet impliceret i patologien. Men vurderer mikroglia funktion in vivo er teknisk udfordrende. Vi har udviklet en enkel men robust teknik til præcis overvågning og kvantificering af fagocytiske potentiale mikroglia i et fysiologisk miljø.

Vurdering Retinal mikrogliaceller fagocytotisk funktion<em&gt; In vivo</em&gt; Brug en flowcytometri-baseret assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

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JoVE Journal

Immunology and Infection

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Vurdering af Innate Sensing af HIV-1 inficerede CD4<sup&gt; +</sup&gt; T-celler ved plasmacytoide dendritiske celler ved hjælp af en<em&gt; Ex vivo</em&gt; Co-dyrkningssystem.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Image-baserede Flowcytometri Teknik at vurdere ændringer i granulocyt Function<em&gt; In vitro</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Flowcytometri-baserede assay for Overvågning af NK cellefunktioner

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

En Flow flowcytometri-baserede cytotoksicitet analyse til vurdering af menneskers NK celleaktivitet

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay til påvisning af Antigen-specifikke T-celle cytolytisk funktion ved hjælp af en Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

En simpel flowcytometri baseret analyse for at bestemme In Vitro antistof afhængig af forbedring af Dengue Virus ved hjælp af Zika Virus rekreationshjem Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

Flerfarvet Flow flowcytometri-baserede kvantificering af mitokondrier og lysosomer i T celler

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

Vurdering af cellulære immunrespons i frugtflue Drosophila melanogaster, ved hjælp af en i Vivo fagocytose Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain

Immunofluorescens farvning ved hjælp af IBA1 og TMEM119 til Mikroglial tæthed, morfologi og perifer myeloid celle infiltration analyse i muse hjernen

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

Måling naturligt erhvervet fagcytose-inducerende antistoffer mod plasmodium falciparum parasitter ved en Flow Cytometri-Baseret Analyse

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...