COLLAGEN

Collagenmaybeusedtocoatglasscoverslipsforthegrowthofepithelial,endothelialandmusclecells,neurons,PC12andCHOcelllines.TypeIcollagenismostoftenusedforcoatingsubstratesforcellculturebecauseitiseasilyobtainablefromrattails.Forshorttermcultures,collagencanbesimplyappliedtoglasscoverslipsandallowedtodry.1.Dilutecollagensolution1:10-1:50with30%ethanolandspreadoversurfaceofsterileglasscoverslip.2.Airdryinatissueculturehood.3.Cellscanbeseededdirectlyonthecollagensurface.4.Collagencoatingpreparedinthiswaytendstodetachfromtheglassinlong-termcultures.

CollagenIVisthemajorconstituentofbasementmembraneandisthereforeamorephysiologicalcoatingforthecultureofmanycelltypes.Forlong-termcultures,collagenIandIVcanbeappliedtoglasscoverslipsbyfirstcoatingtheglasswithpolylysineorpolyornithine.Thisprovidesamorestablecollagencoating.1.Preparepolylysineorpolyornithine(MWof30,000-70,000)at0.1-1mg/mlin0.15Mboratebuffer(pH8.3).Filtersterilize.2.Addenoughsolutiontopooloversurfaceofsterileglasscoverslip.3.Incubate2-24hoursatroomtemperature.4.Aspiratesolutionandwashcoverslips3timeswithwater.5.Poolcollagensolution,100ug/mlinwateroversurfaceofcoverslip.6.Incubate4-16hours.7.Rinseoncewithmediaandseedwithcells.Alternatively,forlong-termcultures,doublelayeredcollagencoatingscanprovideastablecoating.

Alternatively,forlong-termcultures,doublelayeredcollagencoatingscanprovideastablecoating.

1.SpreadacoupleofdropsofsterilecollagenIsolutiononthesterileglasscoverslip.2.Immediatelyneutralizefor2minuteswithammoniumhydroxidevaporsbyplacingthedishofcoverslipsinacovereddishcontainingfilterpaperwetwithconcentratedammoniumhydroxide.Thiswillcausethecollagentogel.3.Washcoverslipstwicewithsterilewater.4.Gentlyspreadacoupleofdropsofcollagenoverthesurfaceofthegelledcollagenandairdry.5.Usewithinafewhoursforcellculture.

Gelatincanalsobeusedforthecultureofsomecelltypesincludingglialcells.1.Dissolve100mggelatinin100mlwater(tripleglassdistilledorRO).2.Autoclavetosterilize.3.Whilehot,thoroughlymixgelatinsolution.4.Addenoughsolutiontopooloversurfaceofsterileglasscoverslip.

5.Chillfor2-24hoursat4oC.6.Removegelatinbyaspirationandaddsterilewater.7.Dishescanbestoredforuptooneweekat4oC.Removewaterimmediatelybeforeuseforcellculture

POLYLYSINEANDPOLYORNITHINENearlyalltypesofcellsadheretothesepolymersofbasicaminoacids.TheyareparticularlyusefulforthecultureofCNSneurons.TheL-orD-isomerscanbeusedforcellattachment,however,theD-isomermaybepreferredbecauseitisnotsubjecttobreakdownbyproteasesreleasedbycells.Preparepolylysineorpolyornithine(MWof30,000-70,000)at0.1-1mg/mlin0.15Mboratebuffer(pH8.3).Filtersterilize.Addenoughsolutiontopooloversurfaceofsterileglasscoverslip.Incubate2-24hoursatroomtemperature.Aspiratesolutionandwashcoverslips3timeswithmediaorPBS.ImmediatelyaddcellsUSPensionorgrowthmedia.

FIBRONECTIN

Fibronectinisanextracellularmatrixconstituentuseforthecultureofendothelialcells,fibroblasts,neuronsandCHOcells.Stocksolutioncanbepreparedbydissolving1mg/mlfibronectininPBS.Filtersterilizeandfreezeinaliquots.Dilutedstocksolutionto50-100ug/mlinbasalmediumorPBS.Addenoughsolutiontopooloversurfaceofsterileglasscoverslip.Incubatefor30-45minatroomtemperature.AspiratetoremovefibronectinandrinsecoverslipswithmediaorPBS.Immediatelyaddcellsuspensionorgrowthmedia.Donotallowcoatingtodry.

LAMININLamininisanextracellularmatrixconstituentusedforthecultureofneurons,epithelialcells,leukocytes,myoblastsandCHOcells.1.Stocksolutioncanbepreparedbydissolving1mg/mllaminininPBS.Filtersterilizeandfreezeinaliquots.2.Dilutedstocksolutionto10-100ug/mlinbasalmediumorPBS.3.Addenoughsolutiontopooloversurfaceofsterileglasscoverslip.4.Incubateseveralhoursatroomtemperature.5.AspiratetoremovelamininandrinsecoverslipswithmediaorPBS.6.Immediatelyaddcellsuspensionorgrowthmedia.Donotallowcoatingtodry.7.Coatingtheglasscoverslipfirstwithpolylysineorpolyornithineandthenlamininmayincreasetheconcentrationoflamininappliedusingthismethod.

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