小鼠腹腔冲洗提取macrophage方法.pdf

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1、小鼠腹腔冲洗提取macrophage方法MOUSEMACROPHAGEEXTRACTION:INTRAPERITONEALLAVAGEMATERIALS10mlsterilesyringes(oneforeachmouse)20gaugeneedles(oneforeachmouse)PhosphateBufferedSaline,pH7.2:PBS1X(GibcoBRLCat.No.20012-027)Isofluraneforanesthetizing70%EthanolforsprayingInstrumentation,a

2、utoclaved:2surgicalscissors,forceps,curledfineforceps50mlconicaltubes,sterile(oneforeachmousegenotype)Macrophagemedia:20%FBSinDMEMhigh-glucose+penicillin:-50mlFetalBovineSerum(ATCCCat.No.30-2020),thawat37Cbeforeadding(20%FBS)-2.5mlPenicillin-Streptomycin(Invitrogen15140-1

3、22),thawat37Cbeforeadding(1%)-197.5mlDMEMhigh-glucose,+L-glu,noNa-pyruvate(GibcoBRLCat.No.11965-092).Thawat37Cbeforeadding-Combineallandfilter.Storeat4C.Note:CanuseFBSorPen-Strepfromanycompany,aslongasithasbeenkeptsterileandyouuseitconsistentlythroughouttheexperiment.Icebu

4、cket6-wellplatesfortissuecultureMice:Allprocedureswillbeperformedinthetissueculturehoods,followingtissueculturepreventiveproceduresMiceinjectedwith1.5mlof4%Thioglycollate4daysprior:CageMouseIDSexDOBGenotypePROTOCOLMacrophageExtraction1.Obtainallmaterials.Autoclaveinstru

5、ments.Preparemediaasspecifiedandkeepat37C.Obtainmice.2.Fill10mlsyringeswithsterilePBS1Xandcapwith20gaugeneedles.Markeachwithmousenumber,genotypeandidentification.Placeonice.Note:AliquotPBSintoa50mlfalcontubewhenfillingsyringes,sothereislesschangeofcontaminationofentirePBSbo

6、ttle.Fillsyringesfrom50mlfalcontube.3.Anesthetizemouseinanesthetizingchamber-Addasmallamountofisofluranetotissuesinbottomofchamber.-Placemouseinchamber.-Watchforbreathingofmouseandtakeoutwhenitslowsdown.Ifmousediesfromanesthesiainthisstep,itwillnotbeaproblem.4.Euthanizemous

7、ebycervicaldislocation-Holdmousebynapeofneckfirmlyandpulltailwithotherhanduntilyoufeelapopandaseparationbetweencraniumandbackbone.5.Sterilizemouse-Placemouseinabeakerwith70%ethanol.-Placemouseintissueculturehoodimmediately.6.Laporotomyincision-Makeasmallincisioninabdominals

8、kinofmouse(useonesetofforcepsandscissors).-Openupmousebypullingskinwithbothhandsto