非磁珠法中性粒提取protocol

Ficoll-右旋糖酐法提取中性粒細胞采集10mL全血于EDTA預處理過的試管中，用不含鈣和鎂離子的1xHBSS緩沖液稀釋之，比例為1:1。采集的樣本必須盡可能早地進行分離。提前另取一50ml離心管，加入與全血量相等體積的Ficoll-Paque溶液。向Ficoll溶液上方緩慢注入稀釋過的全血，注意不要破壞液面。立即600g室溫下9:2減速離心15分鐘（即ACC-9，DEC-2）。離心后可觀察到血漿、外周血單核細胞（PBMC）、Ficoll-Paque溶液和含有粒細胞的紅細胞沉淀四層。前三層棄掉，以獲得第四層紅細胞和中性粒細胞混合物。立即將混合物重懸于1XHBSS緩沖液5ml中，以進行進一步處理。紅細胞和粒細胞懸浮液立即與等量3%右旋糖酐混合，室溫避光孵育30min。收集上層富含中性粒細胞的上清液，棄去沉淀。500g室溫下離心10min。棄上清。最后用3倍全血量的紅細胞裂解緩沖液裂解紅細胞，裂解時間10分鐘。加入等量1xHBSS緩沖液終止裂解。400g室溫下離心10min。離心后棄上清，立即用1ml含10%FBS的RPMI1640完全培養(yǎng)基重懸沉淀，即得中性粒細胞懸浮液。（可選）鏡下對中性粒細胞計數(shù)，以確定后續(xù)實驗方案。（可選）CD45、CD16、CD66b抗體染色后，流式測定中性粒細胞的細胞數(shù)和純度。注：1、 全過程中的1XHBSS可用無菌生理鹽水代替，量是一樣的，這點有文獻佐證。但如果用HBSS要訂購w/oMg2+&Ca2+的，這種相比于生理鹽水來說優(yōu)點是不會刺激中性粒細胞導致其活化（活化的后果是什么也不是特別清楚…炎癥因子表達上調(diào)？）。2、 3%右旋糖酐用無菌生理鹽水配制。3、 （2）、（3）步驟銜接要快，否則分層效果會比較差。4、 （11）和（12）可能是很重要的，因為每一次能提取的細胞量都因?qū)嶒炚`差及個體異質(zhì)性而不同，如果需要嚴格控制共培養(yǎng)細胞數(shù)目比例的話，這一點必不可少。5、 與腫瘤細胞共培養(yǎng)的比例有待摸索。文獻用5x106腫瘤細胞與100x106血小板共培養(yǎng)，而血液中血小板（100-300x109/ml）相對于中性粒細胞（4-10x109/ml）之比例浮動于10-75:1之間，故設(shè)計共培養(yǎng)的比例目前暫定1x、2x、5x、10x、20x，待免疫熒光、細胞凋亡、細胞周期等實驗結(jié)束后再選擇合適比例做后續(xù)實驗。6、 步驟（8）中3倍紅細胞裂解液，這個體積個人覺得可以再少一點，目前臺上一管血8-9ml（實際收不到10ml），如果（8）、（9）都是3倍量，50ml的管子可能撐不住。7、 流式方法：將所取細胞于500g條件下5min離心（最好是減速離心，但實際操作中不減速好像傷害也不大，注意是g不是rpm），棄上清后加入PBS重懸，再同樣條件下離心，反復三次，再棄上清后，用300-400口L1xbindingbuffer（凋亡試劑盒中有這個，實驗室沒有的話用PBS也行）重懸，立刻加入5口LCD45抗體，避光孵育30分鐘。隨即在流式細胞儀中測定純度。如果順便想測凋亡的話，可在CD45避光孵育完后再按凋亡試劑盒步驟加入AnnexinVFITC/PI，避光孵育15分鐘測流式。、簡易提取法：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