Forhightransfectionefficiencyandlowertoxicity,transfectcellsathighdensity.50-80%confluencyisrecommended.
Cellsshouldbeplated18to24hourspriortotransfectionsothatthecelldensityreaches50~80%confluencyatthetimeoftransfection.Completeculturemediumwithserumandantibioticsisfreshlyaddedtoeachwell2hoursbeforetransfection.
Thefollowingprotocolisgivenfortransfectionina24-wellplate,refertoTable1fortransfectioninothercultureformats.
1.Foreachwell,add0.5mlofnormalgrowthmedium(antibioticdoesnotinfluencetheresult)freshly2hoursbeforetransfection.
2.Foreachwell,dilute0.5μgofDNAin50μlofDMEMwithoutseruminatube,andmixgently.
3.Add1μlofNanoFectreagentintoanothertubewith50μlofDMEMwithoutserum,andmixgently.
4.AddNanoFect/DMEMintoDNA/DMEMsolution.Mixbyvortexingfor5-10seconds.
5.Incubatefor~15minutesatroomtemperaturetoallowforNanoFect/DNAcomplexesself-assembly.
6.Addthe100μlNanoFect/DNAmixdrop-wisetothecellsineachwellandhomogenizebygentlyswirlingtheplate.
7.Returntheplatestothecellcultureincubator.
8.Checktransfectionefficiency24to48hoursposttransfection.
Table1.RecommendedAmountsforDifferentCulture
CultureDishSurface | Area(cm2) | CellNumber | MediumVolume(ml) | Plasmid(μg) | NanoFect(μl) | DiluentVolume(μl) |
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96-well | 0.3 | 1-1.5x104 | 0.1 | 0.1 | 0.3 | 10 |
48-well | 1 | 2.5-5x104 | 0.25 | 0.25 | 0.75 | 20 |
24-well | 2 | 0.5-1x105 | 0.5 | 0.5 | 1.5 | 50 |
12-well | 4 | 1-2x105 | 1 | 1 | 3 | 100 |
6-Well/35mm | 9.5 | 2-4x105 | 2 | 2.5 | 7.5 | 200 |
60mm/T25 | 28 | 5-10x105 | 5 | 6-8 | 15-24 | 300 |
100mm/T75 | 79 | 1.5-3x106 | 10 | 15-20 | 40-60 | 500 |
150mm/T150 | 153 | 5-9x106 | 20 | 25-40 | 65-120 | 1000 |
Note:Fordifferentcelltypes,theoptimalratioofNanoFect(μL):DNA(μg)isaround3:1.WerecommendtheNanoFect(μL):DNA(μg)ratioof2:1asastartingpointwhichusuallygivessatisfactorytransfectionefficiencywithinvisiblecytotoxicity,howevertheamountofNanoFectmaybeadjustedfrom2to4μlperμgofDNAdependingonthecelllinetobetransfected.ToensuretheoptimalsizeofNanoFect/DNAcomplexparticles,werecommendusingserum-freeDMEMwithHighGlucosetodiluteDNAandNanoFectReagent.