Name: an optimized procedure for fluorescence-activated cell sorting (facs) isolation of autonomic neural progenitors from visceral organs of fetal mice
Uploaded: 2012-08-17T13:00:00
Description: Watch this Scientific Journal Video about An Optimized Procedure for Fluorescence-activated Cell Sorting FACS Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice at JoVE.com

Forfatterne vil gerne takke Catherine Alford for forslag om celledissociering metoder og Kevin Weller, David Flaherty og Bretagne Matlock for støtte i Flowcytometri delt ressource på Vanderbilt University Medical Center og Melissa A. Musser for kunstnerisk assistance med illustrationer. Vi takker Drs. Jack Mosher og Sean Morrison for rådgivning om gennemførelse isolering af neuronale stamceller. VMC Flowcytometri Delt Resource understøttes af Vanderbilt Ingram Cancer Center (P30 CA68485) og Vanderbilt Digestive Disease Research Center (P30 DK058404). Dette arbejde blev støttet af midler fra US National Institutes of Health tilskud DK064251, DK086594 og DK070219.

1. Fremstillinq af medier (Alle trin udføres i vævskultur hætte) <ol><li> Kombinere følgende: 44 ml L-15 medium, 0,5 ml 100X penicillin / streptomycin (P / S), 0,5 ml 100 mg / ml bovint serumalbumin (BSA), 0,5 ml 1M HEPES, 5 ml vævskulturkvalitet vand. Sørg for at blande BSA og P / S grundigt, før du tilføjer. Dette rumfang bør være tilstrækkelige til dissociation af op til fem forskellige væv typer. Dette medie vil blive anvendt i trin 3.4 til fremstilling quenching opløsninger til anvendelse efter enzym...

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Mus reporter linjer udtrykker fluorescerende reportere bliver bredt tilgængelige via flere bestræbelser i murine genetik samfund 1,8,9. Som et resultat dissociationen fremgangsmåden illustreret her, kan bredt anvendes til isolering af diskrete neuronale undertyper baseret på neurotransmitter-eller receptor ekspressionsmønstre fra enten føtale eller voksen væv. Mens vi har optimeret denne metode baseret på ekspression af en fluorescerende transgen reporter, kan den også anvendes på prøver, der udtry...

The authors middle initials were omitted from the publication of <a href="http://www.jove.com/video/4188">An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice</a>.

Erratum: An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

<table border="1"><tbody><tr><td> Reagens Navn </td><td> Vendor </td><td> Katalog nr. </td><td> Kommentarer </td></tr><tr><td> Accumax </td><td> Sigma (MFR: Innovative Cell Technologies) </td><td> A7089-100ML </td><td> Opbevares frosset i 1 ml alikvoter </td></tr><tr><td> DNase I </td><td> Sigma </td><td> D-4527 </td><td> Opbevaret frosset ved -20 ° C 5 mg / ml i 1xHBSS, (Bruges i Quench, Quench 1:5) </td></tr><tr><td> 10X PBS pH 7,4 </td><td> Gibco </td><td> 70011-044 </td><td> Fyld op til 1 gang med vævskulturkvalitet vand og derpå sterilfilter </td></tr><tr><td> 10X HBSS w / o Ca eller Mg </td><td> Gibco </td><td> 14185-052 </td><td> Fyld op til 1 gang med vævskulturkvalitet vand og derpå sterilfilter </td></tr><tr><td> Leibovitzs L-15 medium </td><td> Gibco </td><td> 21083027 </td><td></td></tr><tr><td> Penicillin / streptomycin 100X </td><td> Gibco </td><td>15140-133 </td><td> Store alikvoteret ved -20 ° C </td></tr><tr><td> BSA </td><td> Sigma </td><td> A3912-100G </td><td> Opbevares alikvoteret ved -20 ° C, 100 mg / ml i vand </td></tr><tr><td> Biowhittaker 1M HEPES i 0,85% NaCl </td><td> Lonza </td><td> 17-737E </td><td></td></tr><tr><td> 38 um Nitex nylonnet membran </td><td> Sefar America </td><td> 3-38/22 </td><td> Skæres i ~ 3 cm firkanter. UV behandling natten over for at sterilisere i vævskultur hætten. </td></tr><tr><td> 7-AAD </td><td> Invitrogen </td><td> A1310 </td><td> 1 mg / ml </td></tr><tr><td> TRIzol LS </td><td> Invitrogen </td><td> 10296-028 </td><td></td></tr><tr><td> 5 ml polystyrenrør </td><td> Falcon </td><td> 352058 </td><td></td></tr><tr><td> 15 ml koniske rør </td><td> Corning </td><td> 430790 </td><td></td></tr><tr><td> Gode ​​dissekere Tang </td><td> Gode ​​videnskabelige instrumenter </td><td> 11251-30 </td><td> Dumont # 5 forcep, Dumoxel, standard spids 0.1x0.06mm </td></tr><tr><td> Dissekere Spoon </td><td> Gode ​​videnskabelige instrumenter </td><td> 10370-18 </td><td></td></tr></tbody></table>

an optimized procedure for fluorescence-activated cell sorting (facs) isolation of autonomic neural progenitors from visceral organs of fetal mice

Under udviklingen crista neuralis (NC)-afledte neuronale progenitorceller migrere væk fra neuralrøret til dannelse autonom ganglier i indre organer såsom tarmen og nedre urinveje. Både under udvikling og i modne væv disse celler er ofte meget spredt over hele væv, så isolation af diskrete populationer ved hjælp af metoder som laser capture mikro-dissektion er vanskelig. De kan imidlertid være umiddelbart visualiseres ved ekspression af fluorescerende reportere drevet fra regulatoriske regioner af neuron-specifikke gener, såsom tyrosinhydroxylase (TH). Beskriver vi en fremgangsmåde optimeret til høje udbytter af levedygtige TH + neuronale progenitorceller fra føtale mus viscerale væv, herunder tarm og nedre urogenital kanal (LUT), baseret på dissociation og fluorescens-aktiveret cellesortering (FACS). Th-genet koder det hastighedsbegrænsende enzym til fremstilling af catecholaminer. Enteriske neuronale forfædre begynde at udtrykke TH durheder deres migration i fosterets tarmen 1 og TH er også til stede i en delmængde af voksne bækken ganglier neuroner 2-4. Den første forekomst af denne slægt, og fordelingen af ​​disse neuroner i andre aspekter af LUT og deres isolation er ikke blevet beskrevet. Neuronale progenitorceller, der udtrykker TH kan let visualiseres ved ekspression af EGFP i mus, der bærer den transgene konstruktion Tg (Th-EGFP) DJ76Gsat/Mmnc 1. Vi afbildet ekspression af dette transgenet i føtale mus for at dokumentere fordelingen af ​​TH +-celler i udviklingen LUT på 15,5 dage efter coitus (DPC), designeret morgenen proppen detektion 0,5 DPC, og observeret, at et undersæt af neuronale progenitorceller i sammenflydningsmidlet bækken ganglier udtrykker EGFP. At isolere LUT TH + neuronale stamceller vi optimeret metoder, der oprindeligt blev anvendt til at oprense crista neuralis stamceller fra føtale mus tarmen 2-6. Tidligere bestræbelser på at isolere NC-afledte befolkninger påberåbtefordøjelse med en cocktail af collagenase og trypsin til opnåelse cellesuspensioner til flowcytometri. I vore hænder disse metoder er produceret cellesuspensioner fra LUT med relativt lav levedygtighed. I betragtning af den allerede lave forekomst af neuronale stamfædre i føtale LUT væv, begiver vi os ud for at optimere dissociationshastigheder metoder, således at celle overlevelse i de sidste dissocieres ville blive øget. Vi fastslået, at blid dissociation i Accumax (Innovative Cell Technologies, Inc.), manuel filtrering, og flow sortering ved lave tryk tillod os at opnå en ensartet større overlevelse (&gt; 70% af den samlede celler) med efterfølgende udbytter på neurale stamceller er tilstrækkeligt til nedstrøms analyse. Fremgangsmåden beskriver vi kan bredt anvendes til at isolere forskellige neuronale populationer fra enten føtale eller væv fra voksne mus.

Watch this Scientific Journal Video about An Optimized Procedure for Fluorescence-activated Cell Sorting FACS Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice at JoVE.com

An Optimized Procedure for Fluorescence-activated Cell Sorting FACS Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

En optimeret fremgangsmåde til oprensning af crista neuralis-afledte neuronale stamceller fra føtale musevæv er beskrevet. Denne fremgangsmåde drager fordel af ekspression fra fluorescerende reportermolekyler alleler at isolere diskrete populationer af fluorescens-aktiveret cellesortering (FACS). Teknikken kan anvendes til at isolere neuronale underpopulationer hele udviklingen og fra voksne væv.

En optimeret Fremgangsmåde til fluorescens-aktiveret cellesortering (FACS) Isolering af autonome Neurale stamceller fra indre organer af føtale Mus

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the ...

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Neuroscience

An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

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