Name: assessment of neuronal viability using fluorescein diacetate-propidium iodide double staining in cerebellar granule neuron culture
Uploaded: 2017-05-10T14:00:00
Description: Watch this Scientific Journal Video about Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture at JoVE.com

Dette arbejde blev støttet af stipendier fra Zhejiang-provinsens naturvidenskabelige institut (LY15H310007), Det Anvendte Forskningsprojekt om Nonprofit Technology of Zhejiang-provinsen (2016C37110), National Natural Science Foundation of China (U1503223, 81673407), Ningbo International Science and Technology Cooperation Project (2014D10019), Ningbo Kommunale Innovation Team af Life Science and Health (2015C110026), Guangdong Provincial International Cooperation Projekt for Videnskab og Teknologi (nr. 2013B051000038), Shenzhen Basic Research Program (JCYJ20160331141459373), Guangdong-Hong Kong Technologies Cooperation Funding Scheme (GHP / 012 / 16GD), Hong Kong Universitets Forskningsråd (15101014), Hongkong Polytechnic University (G-YBGQ) og KC Wong Magna Fund ved Ningbo Universitet.

Alle procedurer fulgte National Institutes of Health (NIH) Guide til pleje og brug af laboratoriedyr (NIH Publications No. 80-23, revideret 1996) og blev godkendt af Institutional Animal Care and Use Committee (IACUC) ved Ningbo University SYXK-2008-0110). 1. Fremstilling af opløsninger og kulturmedier Bemærk: Reagenser og lagre skal fremstilles under sterile forhold. Steriliser ved filtrering ved anvendelse af et filter med en porestørrelse på 0,22 μm. <o...

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	<li>Yu, 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=Indirubin-3-oxime+prevents+H2O2-induced+neuronal+apoptosis+via+concurrently+inhibiting+GSK3beta+and+the+ERK+pathway.">Indirubin-3-oxime prevents H2O2-induced neuronal apoptosis via concurrently inhibiting GSK3beta and the ERK pathway.</a> Cell Mol Neurobiol. , (2016).</li><li>Cui, W., 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=The+anti-cancer+agent+SU4312+unexpectedly+protects+against+MPP(+)+-induced+neurotoxicity+via+selective+and+direct+inhibition+of+neuronal+NOS.">The anti-cancer agent SU4312 unexpectedly protects against MPP(+) -induced neurotoxicity via selective and direct inhibition of neuronal NOS.</a> Br J Pharmacol. 168 (5), 1201-1214 (2013).</li><li>Jebelli, J., Piers, T., Pocock, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+Depletion+of+Microglia+from+Cerebellar+Granule+Cell+Cultures+Using+L-leucine+Methyl+Ester.">Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester.</a> J Vis Exp. (101), e52983 (2015).</li><li>Labno, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Two way to count cells with ImageJ. , (2008).</li><li>Haag, 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=Nos2+Inactivation+promotes+the+development+of+medulloblastoma+in+Ptch1(+/-)+mice+by+deregulation+of+Gap43-dependent+granule+cell+precursor+migration.">Nos2 Inactivation promotes the development of medulloblastoma in Ptch1(+/-) mice by deregulation of Gap43-dependent granule cell precursor migration.</a> Plos Genet. 8 (3), e1002572 (2012).</li><li>Lee, H. Y., Greene, L. A., Mason, C. A., Manzini, M. 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+post-natal+mouse+cerebellar+granule+neuron+progenitor+cells+and+neurons.">Isolation and culture of post-natal mouse cerebellar granule neuron progenitor cells and neurons.</a> J Vis Exp. (23), e990 (2009).</li><li>Messer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+maintenance+and+identification+of+mouse+cerebellar+granule+cells+in+monolayer+culture.">The maintenance and identification of mouse cerebellar granule cells in monolayer culture.</a> Brain Res. 130 (1), 1-12 (1977).</li><li>Li, W., 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=Novel+dimeric+acetylcholinesterase+inhibitor+bis7-tacrine,+but+not+donepezil,+prevents+glutamate-induced+neuronal+apoptosis+by+blocking+N-methyl-D-aspartate+receptors.">Novel dimeric acetylcholinesterase inhibitor bis7-tacrine, but not donepezil, prevents glutamate-induced neuronal apoptosis by blocking N-methyl-D-aspartate receptors.</a> J Biol Chem. 280 (18), 18179-18188 (2005).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat Protoc. 1 (5), 2406-2415 (2006).</li><li>Cheung, G., Cousin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+synaptic+vesicle+pool+replenishment+in+cultured+cerebellar+granule+neurons+using+FM+Dyes.">Quantitative analysis of synaptic vesicle pool replenishment in cultured cerebellar granule neurons using FM Dyes.</a> J Vis Exp. (57), e3143 (2011).</li><li>Atlante, 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=Genistein+and+daidzein+prevent+low+potassium-dependent+apoptosis+of+cerebellar+granule+cells.">Genistein and daidzein prevent low potassium-dependent apoptosis of cerebellar granule cells.</a> Biochem Pharmacol. 79 (5), 758-767 (2010).</li><li>Silva, R., Rodrigues, C., Brites, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+cultured+neuronal+and+glial+cells+respond+differently+to+toxicity+of+unconjugated+bilirubin.">Rat cultured neuronal and glial cells respond differently to toxicity of unconjugated bilirubin.</a> Pediatr Res. 51 (4), 535-541 (2002).</li><li>Jekabsons, M. B., Nicholls, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+analysis+of+cerebellar+granule+neurons+undergoing+apoptosis+by+potassium/serum+deprivation.">Bioenergetic analysis of cerebellar granule neurons undergoing apoptosis by potassium/serum deprivation.</a> Cell Death Differ. 13 (9), 1595-1610 (2006).</li><li>Garner, D. L., Johnson, 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=Viability+Assessment+of+Mammalian+Sperm+Using+Sybr-14+and+Propidium+Iodide.">Viability Assessment of Mammalian Sperm Using Sybr-14 and Propidium Iodide.</a> Biol Reprod. 53 (2), 276-284 (1995).</li></ol>

Denne protokol blev modificeret fra procedurer, der er blevet beskrevet tidligere 6 , 7 . Forskere har brugt tid på at forsøge at reducere ikke-neuronal cellevækst ved at forbedre betingelserne ved dyrkning af primære neuroner in vitro 8 . Men selv med forbedrede kulturbetingelser forbliver nogle glialceller. Desuden er ikke-neuronale celler nødvendige i primære neuronkulturer, fordi de hjælper med neuronal vækst og modni...

Colorimetriske cytotoksicitetsanalyser, såsom 3- (4, 5-dimethyl-2-thiazolyl) -2,5-diphenyl-2-H-tetrazoliumbromid (MTT) assayet, anvendes almindeligvis til at måle cellelevedygtighed in vitro . Primære dyrkede cerebellar granulære neuroner (CGN&#39;er) fra rotter er følsomme for forskellige neurotoksiner, herunder 1-methyl-4-phenylpyridiniumion, hydrogenperoxid og glutamat 1 , 2 . Derfor kan CGN-kulturer anvendes som en in vitro- model inden for neurovidenskab. CGN-kulturer kan indeholde en række celler, herunder neuroner og glialceller, som kan tegne sig for ca. 1% af de samlede celler i CGN-kulturen. Glialceller reagerer imidlertid forskelligt på neurotoksiner sammenlignet med neuroner, hvilket fører til en bias i neuronal levedygtighed målt ved kolorimetriske analyser 3 . I levedygtige celler kan fluoresceindiacetat (FDA) omdannes til fluorescein med esterase.Propidiumiodid (PI) kan interagere med DNA&#39;et efter indtrængning af døde celler og kan bruges til at indikere apoptose i kulturen. Derfor kan FDA-PI dobbeltfarvning samtidig evaluere levedygtige celler og døde celler, hvilket tyder på, at cellelevedygtigheden kan måles mere præcist ved at kombinere begge kolorimetriske metoder. Ved at tilføje Hoechst, en blå fluorescerende plet til kerner, kunne nøjagtigheden af ​​cellelevedygtighed yderligere forbedres. Den her præsenterede protokol beskriver FDA-PI dobbeltfarvning og FDA-PI-Hoechst triple-farvning, som kan bruges til nøjagtigt at analysere neuronal levedygtighed i primære dyrkede CGN&#39;er. Denne protokol udnytter visualisering og skelnen mellem CGN og glialceller i deres forskellige størrelser og former. Efter farvning regnes antallet af levedygtige neuroner og døde neuroner fra repræsentative billeder taget af fluorescerende mikroskopi. De store glialceller er udelukket ved sammenligningen af ​​typiske CGN&#39;er tAken under fluorescerende tilstand med dem taget under fase kontrast mode. En lignende strategi kan udføres for at måle neuronal levedygtighed i blandede cellekulturer indeholdende neuroner og glialceller, såsom primære kortikale kulturer og hippocampalkulturer.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma</td>
			<td>P2636</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytosine &#946;-D-Arabinofuranoside</td>
			<td>Sigma</td>
			<td>C1768</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10099141&#160;</td>
			<td>high quality FBS is essential for culture</td>
		</tr>
		<tr>
			<td>100&#215; glutamine</td>
			<td>Gibco</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>100&#215; anti-biotic</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>BME medium</td>
			<td>Gibco</td>
			<td>21010046&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein diacetate</td>
			<td>Sigma</td>
			<td>F7378</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Yesen</td>
			<td>40731ES10</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit Anti-GAP43 antibody</td>
			<td>Abcam</td>
			<td>ab75810</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse Anti-GFAP antibody</td>
			<td>Cellsignaling</td>
			<td>3670</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sangon Biotech</td>
			<td>A602440</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>Sigma</td>
			<td>T4665</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse&#160;</td>
			<td>Sigma</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T9003</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Volac</td>
			<td>Z310727</td>
			<td>burn&#160; the tip round before use</td>
		</tr>
		<tr>
			<td>12-well cell culture plates</td>
			<td>TPP</td>
			<td>Z707783</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plates</td>
			<td>TPP</td>
			<td>Z707759</td>
			<td>high quality cell culture plate&#160; is essential for culture</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet 5 ml</td>
			<td>Excell Bio</td>
			<td>CS017-0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet 10 ml</td>
			<td>Excell Bio</td>
			<td>CS017-0004</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissect microscope</td>
			<td>Shanghai Caikang</td>
			<td>XTL2400</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Scientific&#160;</td>
			<td>311</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope&#160;</td>
			<td>Nikon</td>
			<td>TI-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence filter and emmision cubes</td>
			<td>Nikon</td>
			<td>B-2A, G-2A, UV-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Photo software</td>
			<td>Nikon</td>
			<td>NIS-Elements</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphics editor softeware</td>
			<td>Adobe</td>
			<td>Photoshop CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Image process softeware</td>
			<td>NIH</td>
			<td>ImageJ</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of neuronal viability using fluorescein diacetate-propidium iodide double staining in cerebellar granule neuron culture

Primære dyrkede cerebellar granulære neuroner (CGN&#39;er) er blevet anvendt i vid udstrækning som en in vitro- model inden for neurovidenskab og neurofarmakologi. Sameksistensen af ​​glialceller og neuroner i CGN-kultur kan imidlertid føre til forstyrrelser i den nøjagtige vurdering af neuronal levedygtighed. Fluoresceindiacetat (FDA) og propidiumiodid (PI) dobbeltfarvning er blevet anvendt til at måle celle-levedygtighed ved samtidig evaluering af de levedygtige og døde celler. Vi anvendte FDA-PI dobbeltfarvning for at forbedre følsomheden af ​​de kolorimetriske analyser og at evaluere neuronal levedygtighed i CGN&#39;er. Derudover tilføjede vi blå fluorescerende DNA-pletter ( f.eks. Hoechst) for at forbedre nøjagtigheden. Denne protokol beskriver hvordan man kan forbedre nøjagtigheden af ​​vurderingen af ​​neuronal levedygtighed ved at anvende disse metoder i CGN-kultur. Ved hjælp af denne protokol kan antallet af glialceller udelukkes ved anvendelse af fluorescensmikroskopi. En lignende strategi kan anvendes til at skelne den uønskede gliAlceller fra neuroner i forskellige blandede cellekulturer, såsom primær kortikultur og hippocampalkultur.

Den dobbelte immunostaining af GAP43 (rød) og GFAP (grøn) blev brugt til at analysere formen af ​​neuroner og glialceller henholdsvis 2 , 5 . Både neuroner og glialceller er til stede i CGN-kultur. De GFAP-positive glialceller er store og uregelmæssige i form, som angivet ved pilene i billederne ( Figur 1 ). Traditionelle assays for celle vitalitet kan ikke skelne glialceller fra neuroner, når de bruges ...

Watch this Scientific Journal Video about Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture at JoVE.com

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Denne protokol beskriver, hvordan man nøjagtigt måler neuronal levedygtighed ved anvendelse af fluoresceindiacetat (FDA) og propidiumiodid (PI) dobbeltfarvning i dyrkede cerebellære granulære neuroner, en primær neuronalkultur, der anvendes som in vitro- model i neurovidenskab og neurofarmakologiforskning.

Vurdering af neuronal levedygtighed ved anvendelse af fluoresceindiacetat-propidiumiodid dobbeltfarvning i cerebellar granulærneuronkultur

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

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