Name: monitoring astrocyte reactivity and proliferation in vitro under ischemic-like conditions
Uploaded: 2017-10-21T13:00:00
Description: Watch this Scientific Journal Video about Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions at JoVE.com

Forfatterne vil takke Paola López Pieraldi for teknisk assistanse. A.H.M. er takknemlig for tilskudd 8G12MD007600 og U54-NS083924 som støttes av denne publikasjonen. Vi takker NIH-NIMHD-G12-MD007583 stipend for anlegget støtte. D.E.R.A. er takknemlig for fellesskap av NIHNIGMS-R25GM110513. Vi er takknemlige for bruk av vanlige instrumentering og ved hjelp av Dr. Priscila Sanabria for bruk av optisk Imaging anlegg av programmet RCMI ved å gi G12MD007583. I tillegg ønsker vi å takke Jose Padilla for sin enestående rolle i filming og redigering visual protokollen.

postnatal rotter (Sprague Dawley) 1-4 dager gamle brukes til å isolere halvdelene. Euthanasia er halshogging, som er godkjent av NIH retningslinjer.
 1. forberedelse av instrumenter og materialer for kirurgi <ol><li>Sterilize instrumenter i en autoklav (temperatur: 121 ºC, Press: 15 psi, tid: 30 minutter) ved hjelp av en stålkasse eller et øyeblikk forsegle sterilisering veske. Se materialer i Tabellen of Materials.</li>
</ol> 2. Fullføre DMEM fo...

<ol>
	<li>Goldstein, L. B., Bertels, C., Davis, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrater+reliability+of+the+NIH+stroke+scale.">Interrater reliability of the NIH stroke scale.</a> Arch Neurol. 46 (6), 660-662 (1989).</li><li>Hinkle, J. L., Guanci, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+ischemic+stroke+review.">Acute ischemic stroke review.</a> J Neurosci Nurs. 39 (5), 285-293 (2007).</li><li>Kassner, A., Merali, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Blood-Brain+Barrier+Disruption+in+Stroke.">Assessment of Blood-Brain Barrier Disruption in Stroke.</a> Stroke. 46 (11), 3310-3315 (2015).</li><li>Moskowitz, M. A., Lo, E. H., Iadecola, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+science+of+stroke:+mechanisms+in+search+of+treatments.">The science of stroke: mechanisms in search of treatments.</a> Neuron. 67 (2), 181-198 (2010).</li><li>Ben Haim, L., Carrillo-de Sauvage, M. A., Ceyzeriat, K., Escartin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elusive+roles+for+reactive+astrocytes+in+neurodegenerative+diseases.">Elusive roles for reactive astrocytes in neurodegenerative diseases.</a> Front Cell Neurosci. 9, 278 (2015).</li><li>Broderick, 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=Guidelines+for+the+Management+of+Spontaneous+Intracerebral+Hemorrhage+in+Adults+2007+Update:+A+Guideline+From+the+American+Heart+Association/American+Stroke+Association+Stroke+Council,+High+Blood+Pressure+Research+Council,+and+the+Quality+of+Care+and+Outcomes+in+Research+Interdisciplinary+Working+Group:+The+American+Academy+of+Neurology+affirms+the+value+of+this+guideline+as+an+educational+tool+for+neurologists.">Guidelines for the Management of Spontaneous Intracerebral Hemorrhage in Adults 2007 Update: A Guideline From the American Heart Association/American Stroke Association Stroke Council, High Blood Pressure Research Council, and the Quality of Care and Outcomes in Research Interdisciplinary Working Group: The American Academy of Neurology affirms the value of this guideline as an educational tool for neurologists.</a> Stroke. 38 (6), 2001-2023 (2007).</li><li>Wang, R., 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=Oxygen-glucose+deprivation+induced+glial+scar-like+change+in+astrocytes.">Oxygen-glucose deprivation induced glial scar-like change in astrocytes.</a> PLoS One. 7 (5), e37574 (2012).</li><li>Sofroniew, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+astrocytes+in+neural+repair+and+protection.">Reactive astrocytes in neural repair and protection.</a> The Neuroscientist. 11 (5), 400-407 (2005).</li><li>Sofroniew, M. V., Vinters, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes:+biology+and+pathology.">Astrocytes: biology and pathology.</a> Acta neuropathologica. 119 (1), 7-35 (2010).</li><li>Souza, D. G., Bellaver, B., Souza, D. O., Quincozes-Santos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+adult+rat+astrocyte+cultures.">Characterization of adult rat astrocyte cultures.</a> PLoS One. 8 (3), e60282 (2013).</li><li>Puschmann, T. B., 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=HB-EGF+affects+astrocyte+morphology,+proliferation,+differentiation,+and+the+expression+of+intermediate+filament+proteins.">HB-EGF affects astrocyte morphology, proliferation, differentiation, and the expression of intermediate filament proteins.</a> J Neurochem. 128 (6), 878-889 (2014).</li><li>Robinson, C., Apgar, C., Shapiro, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+Hypertrophy+Contributes+to+Aberrant+Neurogenesis+after+Traumatic+Brain+Injury.">Astrocyte Hypertrophy Contributes to Aberrant Neurogenesis after Traumatic Brain Injury.</a> Neural Plast. , 1347987 (2016).</li><li>Brekke, E., Berger, H. R., Wideroe, M., Sonnewald, U., Morken, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+and+Intermediary+Metabolism+and+Astrocyte-Neuron+Interactions+Following+Neonatal+Hypoxia-Ischemia+in+Rat.">Glucose and Intermediary Metabolism and Astrocyte-Neuron Interactions Following Neonatal Hypoxia-Ischemia in Rat.</a> Neurochem Res. , (2016).</li><li>Cekanaviciute, E., 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=Astrocytic+transforming+growth+factor-beta+signaling+reduces+subacute+neuroinflammation+after+stroke+in+mice.">Astrocytic transforming growth factor-beta signaling reduces subacute neuroinflammation after stroke in mice.</a> Glia. 62 (8), 1227-1240 (2014).</li><li>Zhu, Z., 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=Inhibiting+cell+cycle+progression+reduces+reactive+astrogliosis+initiated+by+scratch+injury+in+vitro+and+by+cerebral+ischemia+in+vivo.">Inhibiting cell cycle progression reduces reactive astrogliosis initiated by scratch injury in vitro and by cerebral ischemia in vivo.</a> Glia. 55 (5), 546-558 (2007).</li><li>Bovolenta, P., Wandosell, F., Nieto-Sampedro, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+outgrowth+over+resting+and+reactive+astrocytes.">Neurite outgrowth over resting and reactive astrocytes.</a> Restor Neurol Neurosci. 2 (4), 221-228 (1991).</li><li>Anderson, M. 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=Astrocyte+scar+formation+aids+central+nervous+system+axon+regeneration.">Astrocyte scar formation aids central nervous system axon regeneration.</a> Nature. 532 (7598), 195-200 (2016).</li><li>Hao, C., Richardson, A., 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=Macrophage-like+cells+originate+from+neuroepithelium+in+culture:+characterization+and+properties+of+the+macrophage-like+cells.">Macrophage-like cells originate from neuroepithelium in culture: characterization and properties of the macrophage-like cells.</a> Int J Dev Neurosci. 9 (1), 1-14 (1991).</li><li>Saura, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cells+in+astroglial+cultures:+a+cautionary+note.">Microglial cells in astroglial cultures: a cautionary note.</a> J Neuroinflammation. 4, 26 (2007).</li><li>Giulian, D., Baker, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+ameboid+microglia+isolated+from+developing+mammalian+brain.">Characterization of ameboid microglia isolated from developing mammalian brain.</a> J Neurosci. 6 (8), 2163-2178 (1986).</li><li>Schildge, S., Bohrer, C., Beck, K., Schachtrup, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+mouse+cortical+astrocytes.">Isolation and culture of mouse cortical astrocytes.</a> J Vis Exp. (71), (2013).</li><li>Armstrong, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+immature+oligodendrocyte+lineage+cells.">Isolation and characterization of immature oligodendrocyte lineage cells.</a> Methods. 16 (3), 282-292 (1998).</li><li>McCarthy, K. D., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+separate+astroglial+and+oligodendroglial+cell+cultures+from+rat+cerebral+tissue.">Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.</a> J Cell Biol. 85 (3), 890-902 (1980).</li><li>Pont-Lezica, L., Colasse, S., Bessis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+microglia+from+primary+cellular+cultures.">Depletion of microglia from primary cellular cultures.</a> Methods Mol Biol. 1041, 55-61 (2013).</li><li>Svensson, M., Aldskogius, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+density+of+axotomized+hypoglossal+motorneurons+following+pharmacological+blockade+of+the+microglial+cell+proliferation.">Synaptic density of axotomized hypoglossal motorneurons following pharmacological blockade of the microglial cell proliferation.</a> Exp Neurol. 120 (1), 123-131 (1993).</li><li>Wong, V. K., Shapourifar-Tehrani, S., Kitada, S., Choo, P. H., Lee, D. A. <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+rabbit+ocular+fibroblast+proliferation+by+5-fluorouracil+and+cytosine+arabinoside.">Inhibition of rabbit ocular fibroblast proliferation by 5-fluorouracil and cytosine arabinoside.</a> J Ocul Pharmacol. 7 (1), 27-39 (1991).</li><li>Nakatsuji, Y., Miller, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Density+dependent+modulation+of+cell+cycle+protein+expression+in+astrocytes.">Density dependent modulation of cell cycle protein expression in astrocytes.</a> J Neurosci Res. 66 (3), 487-496 (2001).</li><li>Reeves, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+amino+acids+by+lysosomes+incubated+with+amino+acid+methyl+esters.">Accumulation of amino acids by lysosomes incubated with amino acid methyl esters.</a> J Biol Chem. 254 (18), 8914-8921 (1979).</li><li>Thiele, D. L., Kurosaka, M., Lipsky, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotype+of+the+accessory+cell+necessary+for+mitogen-stimulated+T+and+B+cell+responses+in+human+peripheral+blood:+delineation+by+its+sensitivity+to+the+lysosomotropic+agent,+L-leucine+methyl+ester.">Phenotype of the accessory cell necessary for mitogen-stimulated T and B cell responses in human peripheral blood: delineation by its sensitivity to the lysosomotropic agent, L-leucine methyl ester.</a> J Immunol. 131 (5), 2282-2290 (1983).</li><li>Hamby, M. E., Uliasz, T. F., Hewett, S. J., Hewett, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+an+improved+procedure+for+the+removal+of+microglia+from+confluent+monolayers+of+primary+astrocytes.">Characterization of an improved procedure for the removal of microglia from confluent monolayers of primary astrocytes.</a> J Neurosci Methods. 150 (1), 128-137 (2006).</li><li>Corvalan, V., Cole, R., de Vellis, J., Hagiwara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+modulation+of+calcium+channel+activity+in+cultured+rat+astrocytes.">Neuronal modulation of calcium channel activity in cultured rat astrocytes.</a> Proc Natl Acad Sci U S A. 87 (11), 4345-4348 (1990).</li><li>Butler, I. B., Schoonen, M. A., Rickard, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+dissolved+oxygen+from+water:+A+comparison+of+four+common+techniques.">Removal of dissolved oxygen from water: A comparison of four common techniques.</a> Talanta. 41 (2), 211-215 (1994).</li><li>Tasca, C. I., Dal-Cim, T., Cimarosti, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+oxygen-glucose+deprivation+to+study+ischemic+cell+death.">In vitro oxygen-glucose deprivation to study ischemic cell death.</a> Methods Mol Biol. 1254, 197-210 (2015).</li><li>Wu, D., Yotnda, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+testing+of+hypoxia+in+cell+culture.">Induction and testing of hypoxia in cell culture.</a> J Vis Exp. (54), (2011).</li><li>Rivera-Aponte, 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=Hyperglycemia+reduces+functional+expression+of+astrocytic+Kir4.+1+channels+and+glial+glutamate+uptake.">Hyperglycemia reduces functional expression of astrocytic Kir4. 1 channels and glial glutamate uptake.</a> Neuroscience. 310, 216-223 (2015).</li><li>Berger, R., Garnier, Y., Pfeiffer, D., Jensen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharides+do+not+alter+metabolic+disturbances+in+hippocampal+slices+of+fetal+guinea+pigs+after+oxygen-glucose+deprivation.">Lipopolysaccharides do not alter metabolic disturbances in hippocampal slices of fetal guinea pigs after oxygen-glucose deprivation.</a> Pediatric research. 48 (4), 531-535 (2000).</li><li>Anderson, T. R., Jarvis, C. R., Biedermann, A. J., Molnar, C., Andrew, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+the+anoxic+depolarization+protects+without+functional+compromise+following+simulated+stroke+in+cortical+brain+slices.">Blocking the anoxic depolarization protects without functional compromise following simulated stroke in cortical brain slices.</a> Journal of neurophysiology. 93 (2), 963-979 (2005).</li><li>Jarvis, C. R., Anderson, T. R., Andrew, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anoxic+depolarization+mediates+acute+damage+independent+of+glutamate+in+neocortical+brain+slices.">Anoxic depolarization mediates acute damage independent of glutamate in neocortical brain slices.</a> Cerebral Cortex. 11 (3), 249-259 (2001).</li></ol>

Denne protokollen beskriver isolering av astrocyttene fra rotte halvdelene. I denne metoden er det viktig å redusere forurensningen med andre cellulære typer som microglia, oligodendrocytes og fibroblaster. Flere tiltak kan iverksettes for å redusere antall microglia,: endre media, orbital risting og kjemiske behandlinger. Når kultur renhet er bekreftet av immunofluorescence bruke selektiv mobilnettet markører eller fremste celle forurensninger, kan eksperimenter utføres. Antistoff mot ionisert kalsium bindende ada...

Slag er definert som "en akutt Nevrologisk dysfunction av vaskulær opprinnelse med plutselige eller rask utvikling av symptomer og tegn, tilsvarer involvering av fokal områder i hjernen"1,2. Det finnes to typer slag: hemoragisk og iskemiske. Når vaskulær dysfunksjon er forårsaket av aneurisme eller en Arteriovenøs feil, ledsaget av svekkelse med bakre brudd på en arterie, kalles dette hemoragisk hjerneslag3 som, i de fleste tilfeller fører til døden. Når en blodpropp eller en embolus hindrer blodstrøm, kalles forårsaker en midlertidig deprivasjon av oksygen og glukose til en hjernen regionen, det iskemisk slag4. Feil å ernære cellene rundt det berørte området eller iskemisk kjernen fører til en homøostatisk og metabolsk ubalanse, energisk dysfunksjon, neuronal død og betennelse5, som kan indusere en livslang funksjonshemming for pasienter6.
Iskemiske hjerneslag er en multifaktoriell skade som involverer flere typer celler som reagerer og deres effekter på ulike tidspunkt. Mange samhandlinger opprette et vanskelig miljø for å studere atferden til enkeltceller. Så, hvordan vi studere bidrag fra en bestemt celle type under så komplekse miljø? En akseptert i vitro modell av iskemi består av utsette celler til oksygen og glukose deprivasjon (OGD), for en viss periode, etterfulgt av restaurering av celler i et normoxic miljø. Dette systemet simulerer en iskemiske hjerneslag etterfulgt av blod reperfusion. I denne metoden, blir eller vev utsatt for en glukose-frie medier i et miljø renset av oksygen, ved hjelp av en spesialisert hypoxic kammer. OGD inkubasjon tiden kan variere fra noen få minutter til 24 timer, avhengig av hypotesen som ønsker å bli testet. Studier har vist at avhengig av ganger av OGD og normoxic miljø, bestemte fenotyper av hjerneslag (dvs., akutt eller Subkronisk) kan oppnås. Primære isolert astrocyttene, utsatt for OGD med bakre restaurering normoxic forhold, er godt studert mobilnettet modell å etterligne slag i vitro7. Bruke OGD er mulig å avsløre uavhengig molekylære mekanismer av isolerte celler under et slag-lignende miljø.
Som vår kunnskap om astrocytter biologi øker, har det blitt tydelig at de er avgjørende for å opprettholde synapser og opprettholde nevrale reparasjon, utvikling og plastisitet8. Under normale forhold, astrocyttene slipp og svare på cytokiner, chemokines, vekstfaktorer og gliotransmitters, metabolske balanse og homeostase i synapser5,9. I akutt neuroinflammation, for eksempel iskemiske hjerneslag, kan disse cellene bli reaktive, Vis en langsiktig overuttrykte av Glial Fibrillary Sure Protein (GFAP) og Vis hypertrofi i deres morfologi5,10, 11 , 12. som den iskemiske betennelsessykdommer utvikler, homeostase av astrocyttene blir påvirket, om normal glutamat opptak, energi metabolisme, utveksling av aktive molekyler og antioksidant aktivitet13.
Reaktiverte astrocyttene sprer rundt betennelsessykdommer vevet mens leukocytter migrere mot den lesioned område14. Astrocytic spredning kan måles med markører som voksende celle kjernefysiske antigen (PCNA), Ki67 og bromodeoxyuridine (BrdU)15. Denne proliferativ responsen er generert i en tidsavhengige måte og det hjelper danner glial arret, en rekke irreversibelt reaktive astrocyttene langs parenchyma av skadet området etter en skade9. En av de første funksjonene i denne arr er å begrense immun cellen bloduttredelse fra dette området. Men har studier vist at arret blir et fysisk hinder for axons å forlenge, så de slipper molekyler begrenser axonal vekst, og danner en fysisk barriere hindrer axons i å utvide rundt skadde området16. Likevel er det vitenskapelige bevis som viser at etter en ryggmargsskade, fullstendig å hindre glial skjemmende arr kan svekke fornyelse av axons17. Derfor sammenheng der bestemte astrocytic svaret måles, må vurderes på rammen av skaden studerte.
Presentert metodikken kan brukes for å studere individualisert funksjonen av astrocyttene etter glukose oksygenmangel, og det kan endres avhengig av spørsmål som etterforskeren ønsker å svare. For eksempel tillegg morfologiske endringen og markører uttrykt på ulike tidspunkter, OGD, supernatants fra astrocyttene utsatt for OGD kan videre analyseres for å identifisere løselig faktorer utgitt av disse cellene, eller brukes som en betinget media for å vurdere sin effekt i andre hjerneceller. Dette gir studier på astrocytter reaktivitet som kan føre til utviklingen av faktorene som styrer og modulere sine svar i en iskemiske hjerneslag scenario.

<table border="0" cellpadding="0" cellspacing="0" width="933">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:395px;">Instruments for Surgery - Step 1</td>
			<td style="width:167px;"></td>
			<td style="width:119px;"></td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Operating scissor 5.5&#8221;</td>
			<td>Roboz Company</td>
			<td style="width:119px;">RS-6812</td>
			<td style="width:253px;">Tools used to decapitate the rats.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Curved forceps 7&#8221;&#160;</td>
			<td>Roboz Company</td>
			<td>RS-5271</td>
			<td style="width:253px;">Holds the skin of the rat while the skull is removed.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Micro-dissecting scissors 4&#8221;&#160;</td>
			<td>Roboz Company</td>
			<td>RS-5882</td>
			<td style="width:253px;">Cuts both the skin and skull of the rat.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Micro-dissecting forceps 4&#8221; angled, fine sharp&#160;</td>
			<td>Roboz Company</td>
			<td>RS-5095</td>
			<td style="width:253px;">Holds the skin of the rat while the skull is removed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Micro-dissecting forceps 4&#8221; slightly curved 0.8</td>
			<td>Roboz Company</td>
			<td>RS-5135</td>
			<td>Tool used to separate cortices.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Micro-dissecting tweezers</td>
			<td>Roboz Company</td>
			<td>RS-4972</td>
			<td>Peels brain meninges.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Dissection microscope</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:119px;">SZX16</td>
			<td style="width:253px;">Important for removing the meninge from the cortices.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">DMEM Preparation - step 2</td>
			<td style="width:167px;"></td>
			<td style="width:119px;"></td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td>GibCo. Company</td>
			<td>11995-065</td>
			<td style="width:253px;">Supports the growth of cells.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Sodium bicarbonate</td>
			<td>Sigma-Aldrich Company</td>
			<td>S7277</td>
			<td style="width:253px;">Supplement for the cell culture media.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Fetal bovine serum (FBS)</td>
			<td>GibCo. Company</td>
			<td>10437-010</td>
			<td style="width:253px;">Serum-supplement for the cell culture.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Penicillin-Streptomycin&#160;</td>
			<td>GibCo. Company</td>
			<td>15140-148</td>
			<td style="width:253px;">Inhibits the growth of bacterias in the cell culture.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter System 1L with 0.22um pore</td>
			<td>Corning</td>
			<td>431098</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Astrocyte culture - step 3</td>
			<td></td>
			<td></td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Serological pipets 5mL</td>
			<td style="width:167px;">VWR</td>
			<td style="width:119px;">89130-896</td>
			<td style="width:253px;">To pipette DMEM to containers with cells.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Serological pipets 10mL</td>
			<td style="width:167px;">VWR</td>
			<td style="width:119px;">89130-898</td>
			<td style="width:253px;">To pipette DMEM to containers with cells.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Serological pipets 25mL</td>
			<td style="width:167px;">VWR</td>
			<td style="width:119px;">89130-900</td>
			<td style="width:253px;">To pipette DMEM to containers with cells.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Centrifuge conical tube 15mL</td>
			<td style="width:167px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-200250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Safe-lock tube 1.5mL</td>
			<td style="width:167px;">Eppendorf</td>
			<td style="width:119px;">022363204</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Barrier Tips 200 uL</td>
			<td style="width:167px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-201725</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Barrier Tips 1 mL</td>
			<td style="width:167px;">Santa Cruz Biotechnology</td>
			<td>sc-201727</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Biohazard Orange Bag 14 x 19&#34;</td>
			<td>VWR</td>
			<td>14220-048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">60mm petri dishes</td>
			<td>Falcon</td>
			<td>351007</td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Sterile gauze pads</td>
			<td>Honeywell Safety</td>
			<td>89133-086</td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Stomacher 80 Biomaster</td>
			<td style="width:167px;">Sewar Lab System</td>
			<td style="width:119px;">030010019</td>
			<td style="width:253px;">Triturate the brain tissue.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Stomacher 80 Blender Sterile Bags</td>
			<td style="width:167px;">Sewar Lab System</td>
			<td style="width:119px;">BA6040</td>
			<td style="width:253px;">Sterile bag for the stomacher cell homogenizer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Beaker 400mL</td>
			<td style="width:167px;">Pyrex</td>
			<td style="width:119px;">1000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Sterile cell dissociation sieve, mesh #60&#160;</td>
			<td>Sigma-Aldrich Company</td>
			<td>S1020</td>
			<td style="width:253px;">To obtain a uniform single cell suspension.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Sterile cell dissociation sieve, mesh #100</td>
			<td>Sigma-Aldrich Company</td>
			<td>S3895</td>
			<td style="width:253px;">To obtain a uniform single cell suspension.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Invert phase microscope</td>
			<td style="width:167px;">Nikon</td>
			<td style="width:119px;">Eclypse Ti-S</td>
			<td style="width:253px;">Verify cells for contamination or abnormal cell growth.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">75cm2 sterile flasks</td>
			<td>Falcon</td>
			<td>353136</td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Multi-well plate</td>
			<td>Falcon</td>
			<td>353046</td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Micro cover glasses (coverslips), 18mm, round</td>
			<td>VWR</td>
			<td>48380-046</td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Bright-Line hemacytometer</td>
			<td style="width:167px;">Sigma-Aldrich Company</td>
			<td style="width:119px;">Z359629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Pasteur pipettes</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">13-678-20D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Ethyl alcohol&#160;</td>
			<td style="width:167px;">Sigma-Aldrich Company</td>
			<td style="width:119px;">E7023</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">L-leucine methyl ester hydrochloride 98% (LME)</td>
			<td>Sigma-Aldrich Company</td>
			<td>L1002</td>
			<td style="width:253px;">Promotes the elimination of microglia cells in the primary cortical astrocyte cultutre.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytosine &#946;-D-arabinofuranoside (Ara-C)</td>
			<td>Sigma-Aldrich Company</td>
			<td>C1768</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-Lysine Hydrobromide, mol wt 70,000-150,000</td>
			<td>Sigma-Aldrich Company</td>
			<td>P0899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin/EDTA</td>
			<td>GibCo. Company</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue</td>
			<td>Sigma-Aldrich Company</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffer saline (PBS) tablets</td>
			<td>Calbiochem</td>
			<td>524650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Sterile Water</td>
			<td style="width:167px;">Sigma-Aldrich Company</td>
			<td style="width:119px;">W3500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">&#160;OGD Medium Preparation - step 5</td>
			<td style="width:167px;"></td>
			<td style="width:119px;"></td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Centrifuge conical tube 50 mL</td>
			<td style="width:167px;">VWR</td>
			<td style="width:119px;">89039-658</td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle&#8217;s medium-free glucose</td>
			<td>Sigma-Aldrich Company</td>
			<td>D5030</td>
			<td style="width:253px;">Supports the growth of cells.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Sodium bicarbonate</td>
			<td>Sigma-Aldrich Company</td>
			<td>S7277</td>
			<td style="width:253px;">Supplement for the cell culture media.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Penicillin-Streptomycin&#160;</td>
			<td>GibCo. Company</td>
			<td>15140-148</td>
			<td style="width:253px;">Inhibits the growth of bacterias in the cell culture.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">200mM&#160; L-glutamine&#160;</td>
			<td>GibCo. Company</td>
			<td>25030-081</td>
			<td style="width:253px;">Amino acid that supplements the growth of cells.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phospahet buffer saline (PBS) tablets</td>
			<td>Calbiochem</td>
			<td>524650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Filter System 50mL with 0.22um pore</td>
			<td>Corning</td>
			<td>430320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Centrifuge conical tube 50 mL</td>
			<td style="width:167px;">VWR</td>
			<td style="width:119px;">89039-658</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Single Flow Meter&#160;</td>
			<td>Billups-Rothenberg</td>
			<td>SMF3001</td>
			<td style="width:253px;">Measure gas flow in oxygen purge.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hypoxia Incubator Chamber&#160;</td>
			<td>StemCell</td>
			<td>27310</td>
			<td style="width:253px;">Generates a hypoxic environment for the cell culture.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Traceable Dissolved Oxygen Meter</td>
			<td>VWR</td>
			<td>21800-022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">95% N2/ 5% CO2 Gas Mixture</td>
			<td>Linde</td>
			<td></td>
			<td style="width:253px;">Purges the environment of oxygen.</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:395px;">primary astrocyte immunofluorescence - step 6</td>
			<td style="width:167px;"></td>
			<td style="width:119px;"></td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffer saline (PBS) tablets</td>
			<td>Calbiochem</td>
			<td>524650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Formaline Solution Neutral Buffer 10%</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">HT501128</td>
			<td style="width:253px;">Solution used to fix cells.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Methanol&#160;</td>
			<td style="width:167px;">Fisher</td>
			<td style="width:119px;">A4544</td>
			<td style="width:253px;">Solution used to fix cells.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Non-ionic surfactant (Triton X-100)</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">T8787</td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Fetal bovine serum (FBS)</td>
			<td>GibCo. Company</td>
			<td>10437-010</td>
			<td style="width:253px;">Serum-supplement for the cell culture.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Anti-NeuN</td>
			<td>Cell Signaling</td>
			<td>24307</td>
			<td style="width:253px;">Detects mature neurons, serves to validate the astrocytic culture.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-PCNA</td>
			<td>Cell Signaling</td>
			<td>2586</td>
			<td style="width:253px;">Detects proliferating cells.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propidium Iodide (PI)</td>
			<td>Sigma-Aldrich Company</td>
			<td>P4170</td>
			<td style="width:253px;">Apoptosis staining.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Olig1</td>
			<td>Abcam</td>
			<td>AB68105</td>
			<td style="width:253px;">Detects mature oligodendrocytes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Iba1+</td>
			<td>Wako</td>
			<td>016-20001</td>
			<td style="width:253px;">Detects microglial cells.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Anti-GFAP Conjugated with Cy3&#160;</td>
			<td>Sigma-Aldrich Company</td>
			<td>C9205</td>
			<td style="width:253px;">Detects reactive astrocytes in the treated cells.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Alexa Fluor 488</td>
			<td style="width:167px;">Molecular Probe Life Technology</td>
			<td style="width:119px;">A1101</td>
			<td style="width:253px;">Anti-Mouse Secondary Antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Alexa Fluor 555</td>
			<td style="width:167px;">Molecular Probe Life Technology</td>
			<td style="width:119px;">A21428</td>
			<td style="width:253px;">Anti-Rabbit Secondary Antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">4&#8217;,6&#8217;-diamidino-2-phenylindole (DAPI)</td>
			<td style="width:167px;">Sigma-Aldrich Company</td>
			<td style="width:119px;">D9542</td>
			<td style="width:253px;">Nuclear staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Confocal microscope</td>
			<td style="width:167px;">Olympus</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring astrocyte reactivity and proliferation in vitro under ischemic-like conditions

Iskemiske hjerneslag er en kompleks hjerneskade forårsaket av en blodpropp eller embolus hindrer blodtilførsel til deler av hjernen. Dette fører til deprivasjon av oksygen og glukose, som forårsaker energy feil og neuronal død. Etter en iskemiske hjerneslag fornærmelse, astrocyttene bli reaktive og sprer rundt skade området som det utvikler seg. I dette scenariet er det vanskelig å studere bidraget av astrocyttene til hjernen regionen utsatt for ischemia. Derfor presenterer denne artikkelen en metodikk for å studere primære astrocytter reaktivitet og spredning under en i vitro modell av ischemia-lignende miljø, kalt glukose oksygenmangel (OGD). Astrocyttene ble isolert fra 1-4 dagers-gamle neonatal rotter og antall uspesifisert astrocytic celler ble vurdert astrocytter selektiv markør Glial Fibrillary Sure Protein (GFAP) og kjernefysiske flekker. Perioden der astrocyttene utsettes for OGD tilstanden kan tilpasses, samt prosenten av oksygen de utsatt for. Denne fleksibiliteten gjør at forskere til å beskrive varigheten av iskemiske som betingelsen i forskjellige grupper av celler i vitro. Denne artikkelen omhandler tidsrammer av OGD som induserer astrocytter reaktivitet hypertrofisk morfologi og spredning målt ved immunofluorescence med voksende celle kjernefysiske Antigen (PCNA). Foruten spredning, astrocyttene gjennomgå energi og oksidativt stress, og svare på OGD ved å slippe løselig faktorer til celle medium. Dette mediet kan samles og brukes til å analysere effekten av molekyler utgitt av astrocyttene i primære neuronal kulturer uten celle-til-celle. I sammendraget, kan denne primære celle kultur modellen effektivt brukes til å forstå rollen av isolerte astrocyttene ved skade.

En av de største bekymringene primære astrocytic kultur er tilstedeværelsen av andre celler som nerveceller, oligodendrocytes, fibroblaster og microglia. I figur 1, isolerte celler fra rotte halvdelene hadde media endringer hver 3 dager og var enten ubehandlet eller behandlet med lagt LME for 1 h. 24 timer senere, celler var immunostained for GFAP og counterstained med DAPI. Ubehandlet celler viste gjennomsnittlig 39% ikke-GFAP positiv celler, mens LME-beh...

Watch this Scientific Journal Video about Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions at JoVE.com

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Iskemiske hjerneslag er en kompleks hendelse der bidraget av astrocyttene til berørte hjernen regionen utsatt for glukose oksygenmangel (OGD) er vanskelig å studere. Denne artikkelen presenterer en metode for å skaffe isolert astrocyttene og studere deres reaktivitet og spredning under OGD forhold.

Overvåking astrocytter reaktivitet og spredning in Vitro Under iskemiske-lignende forhold

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

Research

JoVE Journal

Neuroscience

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of ...

Conditions Affecting Social Space in Drosophila melanogaster

Conditions Affecting Social Space in Drosophila melanogaster

Forhold som påvirker sosiale rom i<em&gt; Drosophila melanogaster</em

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Den Indirekte Neuron-astrocyte coculture analyse: En<em&gt; In Vitro</em&gt; Oppsett for detaljert undersøkelse av Neuron-gliaceller interaksjoner

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

En protokoll for å måle Cue reaktivitet i en rotte modell av kokain bruk lidelse

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue ...

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Bestemmelse av eksponering av celloverflate og endocytisk proteinprotein i primære astrocytkulturer ved bruk av biotinylering

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

En roman I Vitro Live bildebehandling analysen av astrocytter-mediert fagocytose bruker pH indikator-konjugerte Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

Diffuse Reflectance Spectroscopy: Getting the Capillary Refill Test Under One's Thumb

Diffus refleksjon spektroskopi: Få Capillary raffinere testen Under ens tommelen

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

Levator Auris Longus Forberedelse for undersøkelse av pattedyr nevromuskulær overføring Under spenning klemme forhold

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Sanntid i Vitro overvåking av Odorant reseptor aktivering av en Odorant i damp fase

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition ...

Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

Visualisere Astrocytt morfologi bruke Lucifer Yellow Iontoforese

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, ...