Description
Byutilizingourinnovativeandproprietarylipidconjugationtechnology,LipoD293(Ver.II)isspeciallydesignedandformulatedbyaddingproprietaryenhancersfortransfectingHEK293cellsandothermammaliancells(Figure1).Asa2ndgenerationliposomebasedDNAtransfectionreagent,LipoD293(Ver.II)offersextremelyhightransfectionefficienciesforHEK293relatedcellsaswellasmanymammaliancellswithlesscytotoxicity.LipoD293reagent(Ver.II),upgradedfromitspreviousversionwithrefinedchemistry,is3~4timesmoreefficientingenedelivery.LipoD293reagent(Ver.II),1.0ml,issufficientfor~666transfectionsin24wellplatesor~333transfectionsin6wellplates.
Figure1.ACartoonShowingLipoD293(Ver.II)EnhancedGeneDelivery
Features
-Topchoiceforhard-to-transfectcells
-Exceptionalhightitersofvirusproduction
-EquallygoodforverylongDNAs(upto180kb)
-EquallygoodforsUSPension293cells(e.g.,293F,293H,etc)
-Highlevelsofrecombinantproteinproduction
-Presenceofserumandantibioticsenhancesefficiencyon293cells
-ExceptionalefficiencyforbothsingleDNAtransfectionandmultiDNAsco-transfection
-Veryaffordable
StorageCondition
Storeat4°C.Ifstoredproperly,theproductisstablefor12monthsorlonger.
ComparisonsofTransfectionEfficiencyofLipoD293DNAInVitroTransfectionReagent(Ver.II)withBrandNameProducts
ComparisonoftransfectionefficiencyofLipoD293reagent(Ver.II)vs.lipofectamine2000(L2K)andFugeneHDonHepG2cells.
RightPanel:ComparisonoftransfectionefficiencyofLipoD293(Ver.II)withLipofecatmine2000(L2K),andFugeneHDonHepG2cells.GFPDNA(pEGFP-N3)wastransfectedwithdifferenttransfectionreagentspermanufacturer"sprotocolstoHepG2cell(culturedonCollagenpretreateddishes).GFPpositivecell(%)andfluorescenceintensityweredetectedbypassingthroughFACS48hoursposttransfection
LeftPanel:presenceofserumandantibioticsenhancesLipoD293(Ver.II)efficiencyonHepG2cells.HepG2cell(grownoncollagentreateddishes)wastransfectedwiththreedifferentconditions-------serumandantibioticsfree,presenceof10%serumandantibioticsfollowedbyremoval5hoursposttransfectionandpresenceof10%serumandantibioticswithoutremoval5hoursposttransfection.
ComparisonoftransfectionefficiencyofLipoD293reagent(Ver.II)vs.lipofectamine2000(L2K),TransITandFugene6onCHOcells.
RightPanel:ComparisonoftransfectionefficiencyofLipoD293(Ver.II)withLipofecatmine2000(L2K),TransITandFugene6onCHOcells.DNAsencodingRenillaluciferase(phRL-CMV)andGFP(pEGFP-N3)weretransfectedwithdifferentDNAtransfectionreagentpermanufacturer"sprotocols.RenillaluciferaseactivityandGFPfluorescenceweredetectedwithRenillaAssaySystemandaNikonEclipsefluorescentmicrocopyrespectively24hoursposttransfection.
LeftPanel:Comparisonofprice($/1.0mlvial)ofLipoD293versusthoseofLipofecatmine2000(L2K),TransITandFugene6.Allthepriceswerecollectedfromthemanufacturers"websites.
AcomparisonoftransfectionefficiencyofLipoD293reagentwithlipofectamine2000(L2K)onahard-to-transfectcell,primaryrataorticsmoothmusclecells.TherataorticsmoothmusclecellswerepreparedandtransfectedwithpEGFP-N3byLipoD293reagent(leftpanel)andLipofecatmine2000(L2K,rightpanel)respectivelypermanufacturers"protocols.ThetransfectionefficiencywasevaluatedbydetectingGFPfluorescencewithaNikonEclipse2000microscopy24hoursposttransfection.TheabovepictureswerekindlyprovidedbyDr.NickolaiDulinofSectionofPulmonaryandCriticalCare,UniversityofChicago.
AcomparisonoftransfectionefficiencyofLipoD293reagentwithFugeneHDonhard-to-transfectcell,LNCapcells.TheLNCapcellsweregrownasATCCrecommendedproceduresandco-transfectedwithpBabe-hygro-SSeCKs(1.5ug)andpEGFP-N3(0.5ug)perwell(6wellplate)LipoD293reagent(leftpanel)andFugeneHD(rightpanel)respectivelypermanufacturers"protocols.ThetransfectionefficiencywasevaluatedbydetectingGFPfluorescencewithaNikonEclipse2000microscopy24hoursposttransfection.TheabovepictureswerekindlyprovidedbyDr.LynGaoofRoswellParkCancerInstitute.
TwoexamplesshowingexceptionalefficiencyofLipoD293reagentonhard-to-transfectcellslikeHepG2andSaoS-2cells.HepG2andSaoS-2cellsin95%confluencyweretransfectedwithpEGFP-N3andpSV-?-galactosidaseDNAsrespectivelyinpresenceofserum/antibiotics.Theefficiencywaschecked48hoursposttransfectionbyZeiss510ConfocalMicroscopyand?-galactosidasestainingkitrespectively.
AcomparisonoftransfectionefficiencyofLipoD293reagentwith293fectinonHEK293cells.HEK293cellstransfectedwithpEGFP-C1plasmidusingLipoD293InVitroDNATransfectionReagent(Ver.II)(upperpanel)andthemostpopularbrandproduct293fectinofInvitrogen(lowerpanel).ThecellswerevisualizedbyNikonEclipseFluorescencemicroscopewithDICphaseimaging(leftpanel)andFITCimaging(rightpanel)24hourspost-transfection.
AcomparisonofLipoD293reagentvs.293fectin,XfectandFugene6transfectionreagentsonproteinproductionwithsuspension293Fcells.30mLof293FcellculturedinstandardculturemediumwastransfectedwithpEGFP-6xHisplasmidusingLipoD293InVitroDNATransfectionReagent(Ver.II)(20ugplasmidDNA),293Fectin(30ugplasmidDNA),Xfect(30ugplasmidDNA)andFugene6(30ugplasmidDNA)permanufacturers"standardtransfectionprotocols. GFPfluorescencewasvisualized48hoursposttransfection(leftpanel)withAforLipoD293,Bfor293fectin,CforXfectandDforFugene6. The6xHistaggedGFPproteinwasthenpurifiedviaNi-NTAaffinitycolumn.5ulof1stelutionfractionwasresolvedonSDS-PAGEfollowedbyCoomassieBrilliantBluestaining(rightupperpanelE)withthelane1forLipoD293,lane2forproteinMarker,lane3forXfect,lane4for293fectinandlane5forFugene6. Theproteinyieldwasquantifiedviaspectrometer(rightlowerpanelF).
AcomparisonofLipoD293(Ver.II)andLipofecatmineLTX(LTX)ongenerationofLentivirus(LV).ThreeCDNAswereco-transfectedwithLipoD293(Ver.II)andLipofectamineLTX(LTX)into293FTcells.AGFPvector,pHR-SIN-cppt-CMVEWP,wasusedtodeterminetiterofLV.1x105293Fcellsperwellwasplatedintoa24wellplatefollowedbyadditionofdifferentofamountsofthevectorsupernatant,1microliter(upperpanel)and10microliters(lowerpanel)respectively.5dayslater,thecellswaspassedwithFACS.Thenumbersattheupperrightcornerindicatethepercentageoftransducedcells.ThetitersofLVgeneratedwithLipoD293andL2Kwerequantifiedtobe8x10^6and3x10^6tu/mlrespectively
TechnicalInformation&Datasheet
-LipoD293(Ver.II)ReagentGeneralProtocol&DataSheet
-LipoD293(Ver.II)ReagentShortProtocol
-LipoD293(Ver.II)ReagentProtocolforTransfectingSuspension293andCHOCells
-LipoD293(Ver.II)ReagentforLentivirusProduction
-LipoD293(Ver.II)ReagentforrAAVProduction
-TechnicalNote&TransfectionTips
Torequestafreetrialsample,pleaseCreateAnAccountwithustoenteryourshippingaddressandemailusatorder@Signagen.com
Testimonials
IamabsolutelythrilledwiththeLipoD293transfectionreagent!IcomparedLipoD293toLipofectaminefortransfectionofplasmidstogenerateviralpseudoparticlesandWOW!Irecovered4timesmorevirususingtheLipoD293reagentthanLipofectamine!!ThisiswonderfulnewsformeandmymentorasitmeansthatIdon"thavetospendsomuchtime(andresources)ontransfecting293Tcellstorecovervirusforinvitroexperiments.MymentorwasalsoveryhappythatLipoD293costsmuchlessthanLipofectamine!!Wehavealreadyordered5mlsofLipoD293andIhavenowconvincedmylabmatestoswitchtoLipoD293sinceIgotsuchgoodresultswithit.Thanksformakingsuchagreatproduct!
--------ChristyLavine,Ph.D.,HarvardUniversity
IwantedtoupdateyouonthefreesampleofLipoD293thatyousenttous.IrecentlytransfectedsomePlat-E"sside-by-sidewithLipofectamine2000,andyourproductout-performedit!!!WearealsovalidatingitwithHeLacells,butotherwiseweareVERYhappywithwhatwehaveseenthusfar!Thankyouforsendingusthefreesample,itdefinatelymadetheSALEforus!
--------CatherineGallo,Ph.D.,UniversityofCincinnati
wehavenowtriedtheLipoD293DNAInVitrotransfectionreagenton293FTcells.InthefirstflaskweusedtheprotocolbyInvitrogenprovidedintheVirapowerboxinsert(Virapower,pBABE-GFPplasmid,plusLipofectamine).InthesecondflaskweusedLipoD293(30ul/6mlmedia)withthesameamountsofVirapowerandplasmidasinthefirstflask.Theresultswerestunning:LipoD293gavetwiceasmanytransformantsasLipofectamine2000...
--------ABetaTesterfromWayneStateUniversity
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但是有在转染前，将细胞进行重新传代的情况，这样做的目的在于保持细胞的活性状态。
“转染前将细胞以1.5-4.5X104 cells/well 的量(具体接细胞数请参考表1）接种在孔板中，于37℃， 5%CO2 的条件下进行培养，18-24小时（ sf9细胞为3-4小时）后转染。”
其次要看下你选择单位的规模如何，上海这边的，你可以看下基尔顿生物，原代细胞培养，动物造模，整体课题外包。
我刚开始做转染，悬浮细胞，分别做过表达和敲减，看了很多文献，大都没有提及转染后是用转染的这同一批细胞同时做pcr，wb，cck8，凋亡，细胞周期；还是说这次转染只做pcr或wb，再转染一次做cck8或细胞周期。剩下的功能试验均同前，转染一次做一次？我养的是悬浮细胞，转染后做cck8这些功能试验前需要离心换液吗？跪谢解答！
目标蛋白对细胞有毒性，导致细胞死亡；
转染试剂以及DNA用量信息需要优化，否则对细胞具有伤害；
细胞贴壁转染之后没有正常换液。
建议：考虑对目标蛋白进行截短构建、尝试其他细胞系统；摸索转染试剂以及DNA用量信息，如果转染试剂毒性太大，可以考虑尝试义翘转染试剂sinofection；对转染后的细胞进行换液处理，如果细胞状态感觉不够理想，可以考虑添加一些血清来帮助细胞恢复健康。
以上所有分析、建议的前提是，细胞培养、无菌操作等等都没有问题。祝顺利，加油~
转染分2种，一种是瞬时转染，即转染后让细胞表达目的蛋白后即提取蛋白，提一次蛋白，转染一次，这种方式一般不传代；
另一种转染为稳定转染，转染后加入一定选择压力进行筛选，没有转染的细胞不能存活，只留下转染的细胞，这种情况下可以筛选单个转染细胞，构建稳定表达某一特定蛋白或基因的细胞系。
各位版友求助，
我使用Hek293构建转染模型，瞬转5质粒，用lipo2000做转染体系。
转染48h，发现荧光较强的细胞都在爬片的边缘，比例十分少。是因为我添加试剂的手法不对吗。
同时也发现加入转染体系后细胞状态特别差。想问一下用lipo2000时可以用无双抗的10%FBSDMEM吗。我转染前6h现在用的是纯DMEM，不含FBS。
请各位大神帮忙
不建议你用MDCK细胞，非常难转染。
做的比较好的，一般都是上海地区的，你可以看下基尔顿生物。
