Name: isolation of basal cells and submucosal gland duct cells from mouse trachea
Uploaded: 2012-09-14T12:00:00
Description: Watch this Scientific Journal Video about Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea at JoVE.com

Vi vil gerne anerkende de overordnede Stem Cell Research Center FACS og især takke Jessica Scholes og Felicia Codrea for deres hjælp med cellesortering. Arbejdet blev finansieret af CIRM RN2-00.904-1, K08 HL074229, American Thoracic Society / KOL Foundation ATS-06-065, The Concern Foundation, The UCLA Jonsson Comprehensive Cancer Center Thoracic Oncology Program / Lung Cancer SPORE, University of California Cancer Forskning Samordningsudvalg og Gwynne Hazen Cherry Memorial Laboratories (BG).

Oversigt over Steps 1. Dissektion af trachea 2. Rengøring luftrøret og skære det 3. Enzymfordøjelse og forarbejdning til enkelt cellesuspension 4. Farvning for FACS og sortering 5. Behandling sorterede celler til in vivo-og in vitro-modeller 1. Dissektion af Luftrør <ol><li> Aflive musen med en intraperitoneal injektion af 0,1 mg/0.2 cc...

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Denne teknik til at isolere kanalen og basale celler fra luftvejene er vigtig for at forbedre vores forståelse af luftvejs reparation og regenerering og luftvejs-sygdomme. De beskrevne teknikker her omfatte et par kritiske trin. Den første er den optimeret enzymatisk fordøjelse periode. Den anden er skabelsen af ​​en enkelt celle suspension via seriel passage med progressivt højere gage nåle til at forhindre cell forskydning, men at bryde op celleklumper. Den tredje er FACS-analyse, og gating af celler med egne...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> Komplet medium 10: DMEM-F-12, 50/50, 1X) </td><td> Mediatech </td><td> 15-090-CV </td></tr><tr><td> Hepes (15 mM) </td><td> Invitrogen </td><td> 15.630 </td></tr><tr><td> Natriumbicarbonat (3,6 mM eller 0,03%) </td><td> Invitrogen </td><td> 25.080 </td></tr><tr><td> L-glutamin (4 mM) </td><td> Mediatech </td><td> 25 til 005-CI </td></tr><tr><td> Penicillin (100 U / ml) </td><td> Mediatech </td><td> 30 til 001-CI </td></tr><tr><td> Streptomycin (100 ug / m) </td><td> Mediatech </td><td> 30 til 001-CI </td></tr><tr><td> Amphotericin B (0,25 ug / ml) </td><td> Lonza </td><td> 17-836R </td></tr><tr><td> Iinsulin (10 pg / ml) </td><td> Sigma </td><td> I6634 </td></tr><tr><td> Transferrin (5 ug / ml) </td><td> Sigma </td><td> T1147 </td></tr><tr><td> Choleratoksin (0,1 ug / ml) </td><td> Sigma </td><td> C8052 </td></tr><tr><td> Epidermal vækstfaktor (25 ng / ml) </td><td> BD </td><td> 354.001 </td></tr><tr><td> Ekstrakt fra bovin hypofyse (30 pg / ml) </td><td> Invitrogen </td><td> 13028-014 </td></tr><tr><td> Føtalt bovint serum (5%) </td><td> Fisher </td><td> SH3008803HI </td></tr><tr><td> Retinsyre (0,05 uM) </td><td> Sigma </td><td> R2625 </td></tr><tr><td> Growth Factor Reduceret Matrigel </td><td> BD </td><td> 354.230 </td></tr></tbody></table> Tabel 1. Komplet mediumbestanddele. <table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td&gt; <td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Pronase </td><td> Roche </td><td> 10165921001 </td><td> Bruges til 0,15%: -O / n ved 4 ° C spaltning at isolere total luftrør celler (for ALI kultur) -4 Hr fordøjelse 4 ° C for at isolere SMG </td></tr><tr><td> Dispase </td><td> BD Biosciences </td><td> 354.235 </td><td> Anvendes på 16 enheder: 30 min ved stuetemperatur </td></tr><tr><td> DNase I </td><td> Sigma </td><td> DN25 </td><td> Anvendt ved 0,5 mg / ml: 20-30 min ved stuetemperatur </td></tr></tbody></table> Tabel 2. Enzymer, der anvendes til enzymatisk fordøjelse af luftrøret.

isolation of basal cells and submucosal gland duct cells from mouse trachea

Den store luftveje er direkte i kontakt med miljøet, og derfor modtagelige for skade fra toksiner og smitstoffer, som vi ånde i 1. Den store luftveje kræver derfor en effektiv reparation mekanisme til at beskytte vores kroppe. Denne reparation proces sker fra stamceller i luftveje og isolere disse stamceller fra luftvejene er vigtigt for at forstå mekanismerne af reparation og regenerering. Det er også vigtigt for forståelsen af unormal reparation, kan føre til luftvejssygdomme 2. Målet med denne fremgangsmåde er at isolere en hidtil ukendt stamcellepopulation fra muse tracheale submukøse kirtel kanaler, og at placere disse celler i in vitro og in vivo modelsystemer til at identificere mekanismerne for reparation og regenerering af de submukøse kirtler 3. Denne produktion viser fremgangsmåder, der kan anvendes til isolering og assay kanalen og basale stamceller fra store luftveje 3. Denne måde kan viat undersøge sygdomme i luftvejene, såsom cystisk fibrose, astma og kronisk obstruktiv lungesygdom. I øjeblikket er der ingen fremgangsmåder til isolering af submukøse kirtel kanalen celler, og der er ingen in vivo modeller til at studere regeneration af submukøse kirtler.

Watch this Scientific Journal Video about Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea at JoVE.com

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

Her viser vi vores protokol til isolering af basale og submukøs kirtel kanalen celler fra mus luftrør. Vi demonstrerer også metoden til injektion af stamceller i den dorsale muse fedtpuden at skabe en<em&gt; In vivo</em&gt; Model af submukøse kirtler regenerering.

Isolering af basale celler og submucosal Gland Duct celler fra mus Luftrøret

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

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