Isolation of colonic epithelium

ThemethodweuseisbasedonworkofDr.HazelCheng,attheUniversityofTorontoandworksforbothcolonandthesmallintestine.Firstweexcisethewholecolonfromcecumtorectum.Thisiseasilydoneafteropenningtheabdomenbygraspingthececumwithforcepsandpullinguntilitisstraight.Thencutclosetothececumandtherectum.

2.Flushthecolonusinga10mlsyringeandalargeneedle,preferablywiththepointcutoff,eg.#16.Thisistoremovethecontents,whichyoucanseeflushout.Theflushingsolutionprobablydoesnotmatter.Weuseeithersalineortheincubationsolutionwhichisusedinthe4thstep.

3.Invertthecolon.Thisiseasyonceyouhavedoneitafewtimes,buttrickyatfirst.Thereareseveraldifferenttechniquesthatpeopleuseinthelab.Oneistopassacrochethookordisectingneedlewithahookedendthroughthecolon,andhookontotheoutsideattheotherend.Thenjustdrawtheneedlebackthusturningthecoloninsideout.Asecondwayistoscrunchthecolonontotheendofapairoffineforceptsuntiltheyprotrudefromtheotherend,grabtheendwiththetipoftheforceps(usuallywiththehelpofanotherpair),andthen,again,drawtheforcepsback,turningthecoloninsideout.Actually,Iexpectonecouldsimplyslitthecolonafterflushingbutbecauseoftheworkwedowiththesmallintestine,wearepracticedattheinversion.Possiblythe5thstepwouldbemoredifficultonaslitcolon.

4.Placetheinvertedcolonina0.075MKClsolutioncontaining20mMEDTA(theincubationsolution).Leavefor10-20minutes.Whenthenextstepworkswell,thetimingisright,soyoucantestevery5minutesorso.Seethenextstepforcriteria.

5.Crackthecolon.ThiswasdevelopedbyDavidBlakeyinmylabmanyyearsago.Takea1mltuberculinsyringewithouttheneedle.Puttheendofthesyringeattheendofthecolonandpulltheplungersharply.Thecolonshouldresistbrieflyandthenenterthesyringewithasnap-"crack".Ifthecolonwillnotenter,leaveitlongerintheincubationsolution.Allbutveryoldmousecolonswillworkinourexperience.Ifthecolonenterswithoutasnap,trygrabbingitinthemiddleinsteadoftheend.Ifitstillenterswithoutasnap,reducetheinubationtimeonthenextmouse.Forcethecoloninandoutofthesyringeasfastaspossibleabout5times.Youcanseewhenitisflaccidandthesolutionisnotgettingmoreturbid.Sometimesoneortwocracksareenough.Morethan10areawasteoftime.

6.IsolatetheDNA.Youcanpelletthematerialfirstifyoulike.Ifyouexaminethesuspensionunderthemicroscopeyouwillseeamixtureofcrypts,whichresembleeppindorftubes,brokencrypts,andsinglecells.Ifyoulookafterthefirstcrack,youwillseemostlyintactcrypts,whichisfun.Thereareprobably,almostcertainly,somelymphocytesfromthePeyer"spatchesandsomemyofibroblastsfromthesupportingsubstructurecontaminatingthepreparation,butwearecertainthesearelessthan5%andnormallylessthan1%ofthecells.