fromHoganLab

IsolationofLungBudEndoderm

Whatyouneed:

• E11-12mouseembryos
• DMEMwith5%fetalbovineserum
• petridishesfordissectionsandwashes
• Tyrode-Ringer\"ssolution,pH7.6-7.7(recipebelow)
• pancreatin-trypsinsolution(recipebelow)

1.DissectE11-12lungsinDMEMand5%serumonice.Younglungs(E11to11.5)arebest.

2.Washthelungs3x5minutesoniceinTyrode-Ringer\"sSolution,pH7.6-7.7tooptimizephysiologicalconditionsforpancreatin-trypsinactivity.

3.Incubatewholelungsforappoximately5minutesinpancreatin-trypsinsolutiononice.ThetimerequiredwillvarydependingonthebatchofPT.

4.Washlungs3x5minutesinDMEM+serumtosaturatepancreatin-trypsinactivity.

5.Usingtungstenneedles,dissectoffthemesenchymeandcutoffthebuds.Alternatively,cutoffthebudsfirstandremovethemesenchymesurroundingthem.

6.Placeintomatrigelmatrixandcultureforthreetofivedays(seeaccompanyingprotocol).Isolatedendodermwillnotsurvivewithoutgrowthfactoraddedtotheculturemedium.

Solutions

Tyrode-Ringer\"sSolution,pH7.6-7.7

componentg/liter

• NaCl8.0
• KCl0.3
• NaH₂PO4.5H₂O0.093
• KH₂PO₄0.025
• NaHCO₃1.0
• Glucose2.0

Pancreatin/TrypsinSolution

componentg/20mlfinalconcentration

• pancreatin0.502.5%
• trypsin0.100.5%
• polyvinylpyrrolidone(PVP)0.100.5%
• Tyrode-Ringer\"sSolution20ml

SUSPendbymixingforabout30minutes.Centrifugeat3000XgintheSorvallhangingbucketrotorfor5minutes.Filtersterilizethesupernatantusing.8micronMilliporefilter(s).Storeinaliquotsat-80C.Thisprocessisabigpainandtimeconsuming,mainlybecausethesupernatantdoesnotfilterefficientlyandtheMilliporefiltersmustbeswitchedoften.

CollagenGels

Whatyouneed:

• Collagen,typeIrattail(catno40236,CollaborativeBiomedicalProducts)
• 10XDMEM,lowglucose(catnoD6921,SigmaCellCulture)
• 0.8MNaHCO₃insterilewater
• four-well(1.5cmdiameter/well)Nunctissueculturedishes(catno176740,NalgeNuncInternational)
• culturemedium(forlungbuds)
• tissuecultureincubator

Tissuestobeculturedareplacedinto50uldropsofcollageninfour-wellNuncdishes.Foreachtreatment,makeaseparate50ulaliquotofcollagengelasdescribedintheprotocolbelowandstoreonice.Directlybeforeaddingsamples,addtheNaHCO₃andmixwell.ItisnecessarytoprepareeachcollagengelseparatelybecauseadditionofNaHCO₃makesthecollagenpolymerizequickly.

1.Placecollagen,10XDMEMandonice.Alsochillthefour-welldishesintherfridgebeforeuse.

2.Mix45ulcollagenand5ul10XDMEM.Mixwellonice.Thecollagenwillnotgelifkeptcold.

3.Add1.9ulofNaHCO₃.Mixwell.Thisstepbeginspolymerizationofthecollagengel.

4.Addthe50ulcollagenmixturetoachilledfour-welldish.

5.Firstprepareabedofcollagenandallowittosetat37篊.Afterithaspolymerized,addyoursampletothebedofcollagenandaddcoldunpolymerizedcollageninalayeroverthetopofthesample.

6.Placeat37篊forapproximately10minutestoallowcompletepolymerization.

• Addculturemediumtocoverthebeadofcollagenandtissueandincubatethesampleat37?C.
• Replacemediadaily.