非磁珠法中性粒提取protocol

Ficoll-右旋糖酐法提取中性粒细胞采集10mL全血于EDTA预处理过的试管中，用不含钙和镁离子的1xHBSS缓冲液稀释之，比例为1:1。采集的样本必须尽可能早地进行分离。提前另取一50ml离心管，加入与全血量相等体积的Ficoll-Paque溶液。向Ficoll溶液上方缓慢注入稀释过的全血，注意不要破坏液面。立即600g室温下9:2减速离心15分钟（即ACC-9，DEC-2）。离心后可观察到血浆、外周血单核细胞（PBMC）、Ficoll-Paque溶液和含有粒细胞的红细胞沉淀四层。前三层弃掉，以获得第四层红细胞和中性粒细胞混合物。立即将混合物重悬于1XHBSS缓冲液5ml中，以进行进一步处理。红细胞和粒细胞悬浮液立即与等量3%右旋糖酐混合，室温避光孵育30min。收集上层富含中性粒细胞的上清液，弃去沉淀。500g室温下离心10min。弃上清。最后用3倍全血量的红细胞裂解缓冲液裂解红细胞，裂解时间10分钟。加入等量1xHBSS缓冲液终止裂解。400g室温下离心10min。离心后弃上清，立即用1ml含10%FBS的RPMI1640完全培养基重悬沉淀，即得中性粒细胞悬浮液。（可选）镜下对中性粒细胞计数，以确定后续实验方案。（可选）CD45、CD16、CD66b抗体染色后，流式测定中性粒细胞的细胞数和纯度。注：1、 全过程中的1XHBSS可用无菌生理盐水代替，量是一样的，这点有文献佐证。但如果用HBSS要订购w/oMg2+&Ca2+的，这种相比于生理盐水来说优点是不会刺激中性粒细胞导致其活化（活化的后果是什么也不是特别清楚…炎症因子表达上调？）。2、 3%右旋糖酐用无菌生理盐水配制。3、 （2）、（3）步骤衔接要快，否则分层效果会比较差。4、 （11）和（12）可能是很重要的，因为每一次能提取的细胞量都因实验误差及个体异质性而不同，如果需要严格控制共培养细胞数目比例的话，这一点必不可少。5、 与肿瘤细胞共培养的比例有待摸索。文献用5x106肿瘤细胞与100x106血小板共培养，而血液中血小板（100-300x109/ml）相对于中性粒细胞（4-10x109/ml）之比例浮动于10-75:1之间，故设计共培养的比例目前暂定1x、2x、5x、10x、20x，待免疫荧光、细胞凋亡、细胞周期等实验结束后再选择合适比例做后续实验。6、 步骤（8）中3倍红细胞裂解液，这个体积个人觉得可以再少一点，目前台上一管血8-9ml（实际收不到10ml），如果（8）、（9）都是3倍量，50ml的管子可能撑不住。7、 流式方法：将所取细胞于500g条件下5min离心（最好是减速离心，但实际操作中不减速好像伤害也不大，注意是g不是rpm），弃上清后加入PBS重悬，再同样条件下离心，反复三次，再弃上清后，用300-400口L1xbindingbuffer（凋亡试剂盒中有这个，实验室没有的话用PBS也行）重悬，立刻加入5口LCD45抗体，避光孵育30分钟。随即在流式细胞仪中测定纯度。如果顺便想测凋亡的话，可在CD45避光孵育完后再按凋亡试剂盒步骤加入AnnexinVFITC/PI，避光孵育15分钟测流式。、简易提取法：PROTOCOLNOTE:Allexperimentswereconductedinaccordancewithlocalinstitutionalethicalguidelines.BloodDrawingThroughantecubitalvenipucture,collect14tubesofbloodfromahealthyvolunteerintogreentop(heparinized)tubesandinverteachtubebeforesettlingonice.Collectbloodwithin10-15minofexperimenttoensureoptimalneutrophilyield.NeutrophilIsolationfromWholeBloodWasheachgreentoptubeofbloodwith5mlofPBSwithoutCaandMgtodilutetheblood.Ineachof4x50mlconicaltubes,add15mlofLymphocyteSeparationMedia(LSM)atRT.Usinga18Gneedlemountedona60mlsyringe,carefullylayerthedilutedbloodontotheLSM,creatingasharpLSM-bloodinterface.NOTE:Avoidmixingofthelayersasmuchaspossible.Centrifugeat800xgfor30minat21°Cwithoutbreak.NOTE:Suddenbreakscancausemixingofthedifferentlayers.Observetheerythrocytesandneutrophilssedimentatthebottom.Aspirateanddiscardthetop2layers(plasmaandLSM)makingsuretogetridoftheinterfacebetweentheselayerswhichcontainslymphocytesandmononuclearcells,leavingonlythebottomredlayer.Toeachtube,add20mlofPhosphateBufferSaline(PBS)and20mlof6%Dextransolution.GentlyinverteachtubetomixwiththelayeroferythrocytesandneutrophilsandallowtositatRTfor30min.NOTE:Thiswillallowredbloodcells(RBCs)tosedimentatthebottom.After30min,transfertheneutrophilrichsupernatantintofreshtubesanddiscardtheRBCpellets.Centrifugesupernatantsat450xgat4°Cfor5min.Aftercentrifugation,discardsupernatant.ThepelletwillcontainmostlyneutrophilsandfewRBCs.Preparealysingsolutionbyadding0.5mloflysingbufferto4.5mlofsterilewater.LyseremainingRBCwiththeprepared5mloflysingsolution,transferringallresuspendedpelletsintoonetube.AllowlysisatRTinthedarkfor10min.Centrifugeat450xgat4°Cfor5min.Discardsupernatantandwashpelletwith5mlPBSwithoutCaandMgandcentrifugeagainat450xgat4°Ctogetridofanyremaininglysingsolution.Thepelletobtainedatthispointcontainstheneutrophils.Resuspendthepelletin30mlofcold3%RPMIandputonice.NOTE:3%RPMIismadebysupplementingRPMImediawith3%FetalBovineSerum(FBS)VerifypurityofthesampleusingMethylenebluestaining.>95%ofcellsshouldbegranulocyteswithmulti-lobarnuclei.UseTrypanbluestainingtoverify>95%viabilityofcellsandcountthefinalneutrophilyield.Diluteneutrophilsinicecold3%RPMItoobtainafinalconcentrationof5x106neutrophils/ml.NETsGenerationStimulateneutrophilswith500nMofPMA(per30mlofneutrophilsolution)andincubateona150x25mmflattissueculturedishwith20mmgridfor4hrat37°C5%CO2.ThiswillallowNETosis.After4hrofstimulation,gentlyaspirateanddiscardthemedia,leavingthelayerofNETsandneutrophilsadheredatthebottom.Donotdisruptthislayer.Usingatotalof15mlofcoldPBSwithoutCaandMgperdish,washthebottomofeachdishbypipetting15mlofPBSonthebottomofthedishinordertoliftoffalladherentmaterialfromthebottom.Collectsolutionobtainedfromwashingeachdish(step3.3)ina15mlconicaltubeandcentrifugefor10minat450xgat4°C.Neutrophilsandanyremainingcellswillpelletatthebottom,leavingacell-freeNET-richsupernatant.Dividesupernatantinto1.5mlmicro-centrifugetubesandspinfor10minat18,000xgat4°C.ThiswillallowallDNAtopellet.NOTE:Centrifugationinlargertubesiseasierandlesstimeconsumingifsuchhighspeedsareattainableontheregularcentrifuge,otherwisewillneedtodividesupernatantsinsmallertubesinordertousehighspeedmicrocentrifuge.DiscardsupernatantandresuspendallpelletsobtainedtogetherinicecoldPBStoaconcentrationcorrespondingto2x107neutrophilsper100plofPBS.Thiswillyieldthecell-freeNETstockthatcanbeusedforsubsequentexperiments.MeasureDNAconcentrationinthesampleobtainedusingspectrophotometryoralternateDNAquantificationtool.Anadequateconcentrationinthesampleshouldrangebetween140-180ng/pl.NET-CancerCellStaticAdhesionAssayAdd100plofthepreviouslyobtainedNETstockperwellina96wellflatbottomplateandallowtocoatwellsO/Nat4°Cinthedark.Between12and20hrlater,verifyformationofauniformmonolayerofcellfreeNETsatthebottomofthewellsunderthemicroscope(Figure1).Atthispoint,gentlyaspirateallnon-adherentmaterialoutofthewells,makingsurenottodisrupttheNETmonolayeratthebottom.Add100plof1%BovineSerumAlbumin(BSA)blockingsolutiontoeachwellandleavefor1hatRT.DetachA549cancercellsfromaT-75flaskofusing2mlof0.25%Trypsinsolution.Oncedetached,add10mlofA549mediatotrypsinizedcellsandcentrifugeat450xgat4°Cfor5min.Discardsupernatantandresuspendcellsinmediatoobtainaconcentrationof2x104cancercellsper100plmedia.NOTE:A549cellsweregrownseparately.Briefly,cellswereculturedandmaintainedinDMEMF12mediacontaining10%FBSand1%PenicillinStreptomycinandincubatedat37°C5%CO2.Once70-80%cellconfluencewasreached,theyweredetachedusing0.25%Trypsin-EDTAandresuspendedinthesamemediadescribedabove.StaincancercellsusingCFSEbyadding1plofCFSEpermlofmediaandleavetostainatRTfor10min.Afterstaining,centrifugedownat450xgat4°Cfor5minthendiscardsupernatantandresuspendcellsininitialvolumeofmediatoobtainaconcentrationof2x104cancercellsper100plmedia.After1hrofNETsblocking,gentlyaspirateblockingsolutionandadd2x104cancercellsinmedia,whichisequi