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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DXY721认为：
悬浮细胞和贴壁细胞在转染过程中差别不大，主要差别在于转染后的筛选，当然如果你做的是瞬时转染就不存在筛选的问题了。
其实转染的过程很简单，问题是能不能转的进去的，转染率能有多少，转进去是否可以稳定表达目的蛋白等等。
我们也是用脂质体做悬浮细胞的转染，说明书上都有具体的操作过程，将脂质体和目的基因按比例混合，然后加到细胞悬液里就OK了，说的简单，实际上还是有一些细节要注意的，比如脂质体和目的基因混合的比例，转染的细胞数，细胞的代数，细胞的状态，有的还要求在转染的前一天传代一次，不过不要怕，这些在脂质体说明书上都有明确的说明，按照说明书做就可以了。
jinghuanlv认为：
悬浮细胞和贴壁细胞转染还是有很大不同的。
脂质体转染的原理基于电荷吸引原理，先形成脂质体-DNA复合物，散布在细胞周围，然后通过细胞的内吞作用，将目的基因导入细胞内，而脂质体复合物与贴壁细胞的接触机会比悬浮细胞高出很多倍，所以，脂质体转染时悬浮细胞的转染效率要明显低于贴壁细胞。
我们实验室转染悬浮细胞是用的电穿孔法，目前为止，悬浮细胞转染的最好方法还是电转，我们实验室用的电转仪是Bio-Rad的，使用条件是电压250V，电容975uF，效果不错，不妨一用。
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。
本人研究生
GFP发出绿色荧光的原理是Ca离子进入GFP的beta-barrel结构中引起的特定能级，因此只要这个结构仍然保持着，就可以发出荧光。
由于GFP的beta-barrel结构非常稳定，一些版本的GFP蛋白（如EGFP）甚至能抵抗94C的高温几分钟而不完全变性，因此想在溶液状态下去掉GFP的荧光是很难的，一般需要用光漂白法。
基于其非常稳定的结构，即便细胞被固定了，仍然会有一部分的GFP蛋白保持其构象而发出荧光。此时荧光可能较弱。在荧光显微镜下是有可能看得到的。
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
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。”
