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 gerne takke Paola López Pieraldi for den tekniske bistand. A.H.M. er glad for grants 8G12MD007600 og U54-NS083924, understøttes denne publikation. Vi takker NIH-NIMHD-G12-MD007583 tilskud til faciliteten støtte. D.E.R.A. er taknemmelig for fellowship leveres af NIHNIGMS-R25GM110513. Vi er taknemmelige for anvendelsen af de fælles arealer, instrumentering og ved hjælp af Dr. Priscila Sanabria for brugen af den optiske Imaging facilitet af programmet RCMI ved at give G12MD007583. Hertil kommer, vil vi gerne takke Jose Padilla for hans enestående rolle i filme og redigere den visuelle protokol.

postnatal rotter (Sprague Dawley) 1-4 dage gamle bruges til at isolere cortex. Metoden til aktiv dødshjælp er halshugning, som er godkendt af NIH retningslinjer.
 1. forberedelse af instrumenter og materialer til kirurgi <ol><li>Sterilize instrumenter i en autoklave (temperatur: 121 ºC, pres: 15 psi, tid: 30 min) ved hjælp af en stålkasse eller et øjeblik forsegling sterilisation pose. Se materialerne i Table of Materials.</li>
</ol> 2. Komplet ...

<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. 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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. 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Denne protokol beskriver isolering af astrocytter fra rotte cortex. I denne metode er det afgørende at reducere forurening med andre cellulære typer såsom mikroglia, oligodendrocytes, og fibroblaster. For at reducere antallet af mikroglia, kan der tages flere skridt: ændre media, orbital ryster og kemiske behandlinger. Når kultur renhed er bekræftet ved immunfluorescens benytter selektiv cellulære markører eller for de mest fremtrædende celle forureninger, kan eksperimenter udføres. For eksempel, kan antistof m...

Slagtilfælde er defineret som "en akut neurologisk dysfunktion af vaskulær oprindelse med enten pludselig eller hurtig udvikling af symptomer og tegn, svarende til inddragelse af fokale områder i hjernen"1,2. Der er to typer af slagtilfælde: blødende og iskæmisk. Når vaskulær dysfunktion er forårsaget enten af en aneurisme eller en arteriovenøs funktionsfejl, ledsaget af svækkelse med posterior ruptur af en arterie, er dette kaldes blødende stroke3 som i de fleste tilfælde fører til døden. Når en blodprop eller en embolus hindrer blodgennemstrømningen, kaldes forårsager en midlertidig frihedsberøvelse af ilt og glucose til et område af hjernen, det iskæmisk slagtilfælde4. Undladelse af at nære cellerne omkring det berørte område eller iskæmiske kerne fører til En homeostatiske og metabolisk ubalance, energiske dysfunktion, neuronal død og inflammation5, som kan fremkalde et livslang handicap for patienter6.
Iskæmisk slagtilfælde er en multifaktoriel skade, som omfatter flere typer af celler, der reagerer og udøve deres virkninger på forskellige tidspunkter. Mange interaktioner oprette et vanskeligt miljø for at studere funktionsmåden for individuelle celler. Så hvordan vi studere bidrag af en bestemt celletype under sådan et komplekst miljø? En accepteret i vitro model af iskæmi består af udsætter celler til ilt og glucose afsavn (OGD), i en vis periode, efterfulgt af restaureringen af celler til et normoxic miljø. Dette system simulerer en iskæmisk slagtilfælde efterfulgt af blod reperfusion. I denne metode, er celler eller væv udsat for en glucose-gratis medier i et miljø, der er renset for ilt, ved hjælp af en specialiseret hypoksiske kammer. OGD inkubationstiden kan variere fra et par minutter op til 24 timer, afhængigt af den hypotese, at ønsker at blive testet. Undersøgelser har vist, at afhængigt af tider af OGD og normoxic miljø, specifikke fænotyper af slagtilfælde (dvs., akut eller subkronisk) kan opnås. Primære isolerede astrocytter, udsat for OGD med posterior restaurering normoxic betingelser, er velundersøgte cellulære model at efterligne slagtilfælde in vitro-7. Ved hjælp af OGD er muligt at afsløre de uafhængige molekylære mekanismer i isolerede celler under et slagtilfælde-lignende miljø.
Vores viden om astrocyte biologi stiger, er det blevet tydeligt, at de er afgørende for at opretholde synapser og fastholde neurale reparation, udvikling og plasticitet8. Under normale omstændigheder, astrocytter frigive og reagere på cytokiner, kemokiner, vækstfaktorer og gliotransmitters, at holde stofskiftebalance og homøostase i synapser5,9. I akutte neuroinflammation, såsom iskæmisk slagtilfælde, kan disse celler blive reaktiv, vise en langsigtet overekspression af Glial fibrillære sure Protein (GFAP), og vise hypertrofi i deres morfologi5,10, 11 , 12. som den iskæmisk infarkt udvikler, homøostase, som astrocytter bliver påvirket, om normale glutamat optagelse, energimetabolisme, udveksling af aktive molekyler, og antioxidant aktivitet13.
Reaktiveret astrocytter formere sig omkring infarkt væv mens leukocytter vandrer mod lesioned område14. Astrocytic spredning kan måles ved hjælp af markører som prolifererende celle nukleare antigen (PCNA), Ki67 og bromodeoxyuridine (BrdU)15. Denne proliferativ svar er genereret i en tidsafhængig måde og det hjælper danner glial arret, en bred vifte af uigenkaldeligt reaktive astrocytter langs parenkym af den skadede lokalitet efter en skade9. En af de oprindelige funktioner i dette ar er at begrænse den immun celle ekstravasation fra dette område. Undersøgelser har dog vist, at arret bliver en fysisk hindring for axoner udvide, som de frigive molekyler hæmme cytoskeletale vækst, og skabe en fysisk barriere, der forhindrer axoner fra strækker sig rundt om det skadede område16. Der er dog videnskabelige beviser for, at efter en rygmarvsskade, helt forhindrer glial ar dannelse kan forringe regenerering af axoner17. Således, den kontekst hvor den specifikke astrocytic reaktion måles, skal tages i betragtning ved rammerne af den skade, der er undersøgt.
Den præsenteres metode kan anvendes til at studere den individualiserede funktion af astrocytter efter glukose iltmangel og det kan ændres afhængigt af de spørgsmål, som efterforsker ønsker at besvare. For eksempel, udover de morfologiske forandringer og markører udtrykt på forskellige OGD tidspunkter, analysere fra astrocytter udsat for OGD kan yderligere analyseres for at identificere opløselige faktorer udgivet af disse celler, eller bruges som konditioneret medie til at vurdere sin effekt i andre hjerneceller. Denne tilgang gør det muligt for undersøgelser på astrocyte reaktivitet, der kan føre til udredning af de faktorer, der styrer og modulere deres svar i en iskæmisk slagtilfælde scenario.

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			<td height="34" style="height:34px;width:395px;">Instruments for Surgery - Step 1</td>
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			<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>
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			<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>
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			<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

Iskæmisk slagtilfælde er en kompleks hjerneskade forårsaget af en blodprop eller embolus blokerer blodtilførslen til dele af hjernen. Dette fører til fratagelse af ilt og glukose, som forårsager energi fiasko og neuronal død. Efter en iskæmisk slagtilfælde fornærmelse, astrocytter blive reaktive og formere sig omkring skaden site, da det udvikler. Under dette scenario er det svært at studere astrocytter til regionen hjernen udsat for iskæmi særlige bidrag. Derfor, denne artikel introducerer en metode til at studere primære astrocyte reaktivitet og spredning under en in vitro- model af en iskæmi-lignende miljø, kaldet glucose iltmangel (OGD). Astrocytter blev isoleret fra 1-4 dag-gamle neonatal rotter og antallet af ikke-specifik astrocytic celler blev vurderet ved hjælp af astrocyte selektive markør Glial fibrillære sure Protein (GFAP) og nukleare farvning. Perioden hvor underkastes astrocytter OGD betingelse kan tilpasses, samt procentdelen af ilt de udsættes for. Denne fleksibilitet giver forskerne til at karakterisere varigheden af iskæmisk-lignende tilstand i forskellige grupper af celler in vitro. I denne artikel beskrives de tidsrammer af OGD, der inducerer astrocyte reaktivitet, hypertrofisk morfologi og spredning målt ved immunfluorescens ved hjælp af prolifererende celle nukleare Antigen (PCNA). Udover spredning, astrocytter gennemgå energi og oxidativ stress, og reagere på OGD ved at frigive opløselige faktorer i celle medium. Dette medie kan indsamles og bruges til at analysere virkningerne af molekyler udgivet af astrocytter i primære neuronal kulturer uden celle til celle interaktion. I sammendrag, kan denne primære celle kultur model bruges effektivt at forstå rollen af isolerede astrocytter efter skade.

En af de største bekymringer af primære astrocytic kultur er tilstedeværelsen af andre celler som neuroner, oligodendrocytes, fibroblaster og mikroglia. I figur 1, isolerede celler fra rotte cortex havde medieÃ¦ndringer hver 3 dag og var enten ubehandlet eller behandlet med tilføjet LME for 1 h. 24 timer senere, celler blev immunostained for GFAP og counterstained med DAPI. Ubehandlede celler viste et gennemsnit på 39% ikke-GFAP positive celler, mens L...

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

Iskæmisk slagtilfælde er en kompleks event hvor astrocytter til regionen berørte hjerne særlige bidrag udsat for glucose iltmangel (OGD) er vanskelige at undersøge. Denne artikel introducerer en metode til at opnå isolerede astrocytter og studere deres reaktivitet og spredning OGD betingelser.

Overvågning Astrocyte reaktivitet og spredning in Vitro iskæmisk-lignende betingelser

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, der påvirker sociale rum 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-astrocyt cokultur assay: En<em&gt; In vitro</em&gt; Opsætning for detaljeret undersøgelse af Neuron-glia Interaktioner

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 protokol til måling af Cue reaktivitet i en rotte Model af kokain brug 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 af celleoverfladeekspression og endocytisk proteinhastighed i primære astrocytkulturer ved anvendelse af 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 In Vitro Live-imaging analyse af Astrocyte-medieret fagocytose bruger pH indikator-konjugeret 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

Diffuse Reflektionsgraden spektroskopi: Kom kapillær Refill Test Under sin tommelfinger

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 til eksamen mammale neuromuskulære transmission spænding klemme betingelser

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

Real-time i Vitro overvågning af lugtstof Receptor aktivering af en lugtstof i fasen for damp

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

Visualisering af Astrocyt morfologi ved hjælp af Lucifer Yellow iontophoresis

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